Methods for solid tumor treatment

ABSTRACT

Disclosed herein are methods for treating solid tumors by direct injection into the tumors of chemotherapeutic particles, methods for inhibiting tumor metastasis by administering chemotherapeutic particles to a subject having a tumor, and compositions that include chemotherapeutic particles, small, amounts of a polysorbate, and a carrier.

CROSS REFERENCE

This application is a continuation of International Application No.PCT/US2017/025718, filed on Apr. 3, 2017, which claims priority to U.S.Provisional Application No. 62/318,014, filed Apr. 4, 2016; and U.S.Provisional Application No. 62/378,543, filed Aug. 23, 2016, all ofwhich are incorporated by reference herein in their entirety.

BACKGROUND

Millions of patients are diagnosed each year world-wide as havingcancer, and millions more die from cancer or cancer-relatedcomplications each year. The risk of cancer increases significantly withage, many cancers occur more commonly in developed countries, and cancerrates are increasing as life expectancy increases in the developedworld. Current therapies include systemic treatments such as intravenous(IV) infusion injection of chemotherapeutic agents. These therapies,however, generally have significant undesired side effects to thepatient due to systemic toxicity. Direct injection of chemotherapeuticagents into tumors has been attempted, however, the chemotherapeuticagents tend to “leak” out of the tumor especially when thechemotherapeutic agents are solubilized. Thus, improved methods fortreating patients having cancer are needed.

SUMMARY OF THE INVENTION

In one aspect of the invention, disclosed is a method for treating asolid tumor, comprising administering to a subject with a solid tumor anamount effective of a composition comprising chemotherapeutic particlesto treat the tumor, wherein the composition is directly injected intothe tumor.

In another aspect of the invention, disclosed is a method for inhibitingtumor metastasis, comprising administering to a subject with a tumor anamount effective of a composition comprising chemotherapeutic particlesto inhibit tumor metastasis.

In another aspect of the invention, disclosed is a suspension comprisingchemotherapeutic particles, a pharmaceutically acceptable carrier, and apolysorbate.

In another aspect of the invention, disclosed is a kit comprising afirst vial containing chemotherapeutic particles; a second vialcontaining a polysorbate; and instructions for reconstituting thechemotherapeutic particles into a suspension and for diluting thesuspension with a diluent solution prior to administration to a patient.

Also disclosed in the context of the present invention are the followingembodiments 1 to 77:

Embodiment 1 is a method for treating a solid tumor, comprisingadministering to a subject with a solid tumor an amount effective of acomposition comprising chemotherapeutic particles to treat the tumor,wherein the composition is directly injected into the tumor, such as amalignant tumor.

Embodiment 2 is the method of embodiment 1 wherein the compositionconsists of the chemotherapeutic particles and a pharmaceuticallyacceptable carrier, such as a liquid carrier.

Embodiment 3 is the method of any one of embodiments 1-2, wherein theadministering results in chemotherapeutic migrating into the lymphaticsystem of the subject.

Embodiment 4 is a method for inhibiting tumor metastasis, comprisingadministering to a subject with a malignant tumor an amount effective ofa composition comprising chemotherapeutic particles to inhibit tumormetastasis.

Embodiment 5 is the method of embodiment 4, wherein the composition isdirectly injected into the tumor, or is peritumorally injected.

Embodiment 6 is the method of any one of embodiments 4-5, wherein thecomposition consists of the chemotherapeutic particles and a carrier(such as a liquid, semi-solid, or solid carrier).

Embodiment 7 is the method of any one of embodiments 1-3 and 6, whereinthe carrier is an aqueous liquid carrier.

Embodiment 8 is the method of embodiment 7 wherein the aqueous liquidcarrier is saline, such as normal saline.

Embodiment 9 is the method of any one of embodiments 1-8, wherein thecomposition is a suspension.

Embodiment 10 is the method of any one of embodiments 1-9 wherein theparticles are (i) uncoated; (ii) not embedded, contained, enclosed orencapsulated within a solid excipient; and (iii) not microspheres,liposomes, or microcapsules containing chemotherapeutic and anexcipient.

Embodiment 11 is the method of any one of embodiments 1-10, wherein thechemotherapeutic is selected from the group consisting of paclitaxel;derivatives of paclitaxel, docetaxel, cabazitaxel, taxanes; epithilones,Vinca alkaloids, such as vinblastine, vincristine, vindesine,vinorelbine; camptothecin analogs; epipodophyllotoxins, such ascisplatin, carboplatin, oxaliplatin, etoposide and teniposide;doxorubicin, anthrcyclines, 5-fluorouracil, topotecan, gemcitabine,peroxisome proliferator-activated receptor (PPAR) ligands, andantiangiogenics, or a pharmaceutically acceptable salt thereof.

Embodiment 12 is the method of any one of embodiments 1-11, wherein thechemotherapeutic is a taxane, or a pharmaceutically acceptable saltthereof.

Embodiment 13 is the method of any one of embodiments 1-12, wherein thechemotherapeutic is paclitaxel, or a pharmaceutically acceptable saltthereof.

Embodiment 14 is the method of any one of embodiments 1-13, wherein thetumor is selected from the group consisting of sarcomas, carcinomas, andlymphomas, breast tumors, prostate tumors, head and neck tumors,glioblastomas, bladder tumors, pancreatic tumors, liver tumors, ovariantumors, colorectal tumors, cutaneous, lymphoid, and gastrointestinaltumors.

Embodiment 15 is the method of embodiment 13, wherein the tumor isselected from the group consisting of ovarian, bladder, breast,prostate, pulmonary, pancreatic, cutaneous, lymphoid, andgastrointestinal tumors.

Embodiment 16 is the method of embodiment 13, wherein the tumor isselected from the group consisting of ovarian and bladder tumors.

Embodiment 17 is the method of any one of embodiments 1-12, wherein thechemotherapeutic is docetaxel, or a pharmaceutically acceptable saltthereof.

Embodiment 18 is the method of embodiment 17, wherein the tumor isselected from the group consisting of ovarian, bladder, breast, andprostate tumors.

Embodiment 19 is the method of embodiment 17, wherein the tumor isselected from the group consisting of breast and prostate tumors.

Embodiment 20 is the method of any one of embodiments 1-19, wherein thechemotherapeutic particles comprise at least 95% chemotherapeutic andwherein the particles have a specific surface area (SSA) of at least 10m²/g, or at least 12 m²/g, 14 m²/g, 16 m²/g, 18 m²/g, 20 m²/g, 25 m²/g,30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

Embodiment 21 is the method of embodiment 20, wherein thechemotherapeutic particles have an SSA of between about 10 m²/g andabout 50 m²/g.

Embodiment 22 is the method of any one of embodiments 20-21, wherein thechemotherapeutic particles have an SSA of:

(a) between 16 m²/g and 31 m²/g or between 32 m²/g and 50 m²/g;

(b) between 16 m²/g and 30 m²/g or between 32 m²/g and 50 m²/g;

(c) between 16 m²/g and 29 m²/g or between 32 m²/g and 50 m²/g;

(d) between 17 m²/g and 31 m²/g or between 32 m²/g and 50 m²/g;

(e) between 17 m²/g and 30 m²/g or between 32 m²/g and 50 m²/g;

(f) between 17 m²/g and 29 m²/g, or between 32 m²/g and 50 m²/g;

(g) between 16 m²/g and 31 m²/g or between 33 m²/g and 50 m²/g;

(h) between 16 m²/g and 30 m²/g or between 33 m²/g and 50 m²/g;

(i) between 16 m²/g and 29 m²/g or between 33 m²/g and 50 m²/g;

(j) between 17 m²/g and 31 m²/g or between 33 m²/g and 50 m²/g;

(k) between 17 m²/g and 30 m²/g or between 33 m²/g and 50 m²/g;

(l) between 17 m²/g and 29 m²/g, or between 33 m²/g and 50 m²/g;

(m) between 16 m²/g and 31 m²/g, or ≥32 m²/g;

(h) between 17 m²/g and 31 m²/g, or ≥32 m²/g;

(i) between 16 m²/g and 30 m²/g, or ≥32 m²/g;

(j) between 17 m²/g and 30 m²/g, or ≥32 m²/g;

(k) between 16 m²/g and 29 m²/g, or ≥32 m²/g;

(1) between 17 m²/g and 29 m²/g, or ≥32 m²/g;

(m) between 16 m²/g and 31 m²/g, or ≥33 m²/g;

(n) between 17 m²/g and 31 m²/g, or ≥33 m²/g;

(o) between 16 m²/g and 30 m²/g, or ≥33 m²/g;

(p) between 17 m²/g and 30 m²/g, or ≥33 m²/g;

(q) between 16 m²/g and 29 m²/g, or ≥33 m²/g; or

(r) between 17 m²/g and 29 m²/g, or ≥33 m²/g.

Embodiment 23 is the method of any one of embodiments 20-22, wherein thechemotherapeutic is paclitaxel, or a pharmaceutically acceptable saltthereof.

Embodiment 24 is the method of any one of embodiments 20-22, wherein thechemotherapeutic is docetaxel, or a pharmaceutically acceptable saltthereof.

Embodiment 25 is the method of any one of embodiments 1-24, wherein thechemotherapeutic particles include at least 95% by weight of a taxane,or a pharmaceutically acceptable salt thereof, wherein the particleshave one or both of the following characteristics:

(i) a mean bulk density between about 0.050 g/cm³ and about 0.15 g/cm³,and/or

(ii) have a specific surface area (SSA) of at least 18 m²/g, 20 m²/g, 25m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

Embodiment 26 is the method of embodiment 25, wherein the taxane isselected from the group consisting of paclitaxel, docetaxel,cabazitaxel, taxadiene, baccatin III, taxchinin A, brevifoliol, andtaxuspine D, or a pharmaceutically acceptable salt thereof.

Embodiment 27 is the method of embodiment 25, wherein the taxane isselected from the group consisting of paclitaxel, docetaxel, andcabazitaxel, or a pharmaceutically acceptable salt thereof.

Embodiment 28 is the method of embodiment 27, wherein the taxane ispaclitaxel or a pharmaceutically acceptable salt thereof, and whereinthe particles have a mean bulk density between about 0.050 g/cm³ andabout 0.12 g/cm³, or between about 0.060 g/cm³ and about 0.11 g/cm³.

Embodiment 29 is the method of embodiment 27 or 28, wherein the taxaneis paclitaxel or a pharmaceutically acceptable salt thereof, and whereinthe paclitaxel particles have a specific surface area (SSA) of at least18 m²/g, 20 m²/g, 25 m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

Embodiment 30 is the method of any of embodiments 27-29, wherein thetaxane is paclitaxel or a pharmaceutically acceptable salt thereof, andwherein the wherein the paclitaxel particles have a SSA of between about22 m²/g and about 40 m²/g, 25 m²/g and about 40 m²/g, 30 m²/g and about40 m²/g, or between about 35 m²/g and about 40 m²/g.

Embodiment 31 is the method of any one of embodiments 28-30, wherein thepaclitaxel particles have a bulk density of between about 0.060 g/cm³and about 0.11 g/cm³ and a SSA of between about 22 m²/g and about 40m²/g.

Embodiment 32 is the method of any one of embodiments 28-31, wherein atleast 40% (w/w) of the paclitaxel is dissolved in 30 minutes or less ina solution of 50% methanol/50% water (v/v) at 37° C. and pH 7.0 in a USPII paddle apparatus operating at 75 RPM.

Embodiment 33 is the method of embodiment 27, wherein the taxane isdocetaxel or a pharmaceutically acceptable salt thereof, and wherein theparticles have a mean bulk density between about 0.050 g/cm³ and about0.12 g/cm³, or between about 0.06 g/cm³ and about 0.1 g/cm³.

Embodiment 34 is the method of embodiment 27 or 33, wherein the taxaneis docetaxel or a pharmaceutically acceptable salt thereof, and whereinthe docetaxel particles have a SSA of at least 18 m²/g, 20 m²/g, 25m²/g, 30 m²/g, 35 m²/g, 40 m²/g, or 42 m²/g.

Embodiment 35 is the method of embodiment 27 or 33-34, wherein thetaxane is docetaxel or a pharmaceutically acceptable salt thereof, andwherein the docetaxel particles have a SSA of between about 40 m²/g andabout 50 m²/g, or between about 43 m²/g and about 46 m²/g.

Embodiment 36 is the method of any one of embodiments 33-35, wherein thedocetaxel particles have a bulk density of between about 0.06 g/cm³ andabout 0.1 g/cm³ and a SSA of between about 40 m²/g and about 50 m²/g.

Embodiment 37 is the method of any one of embodiments 33-36, wherein atleast 20% (w/w) of the docetaxel is dissolved in 30 minutes or less in asolution of 15% methanol/85% water (v/v) at 37° C. and pH 7.0 in a USPII paddle apparatus operating at 75 RPM.

Embodiment 38 is the method of any one of embodiments 1-22, wherein thechemotherapeutic particles include at least 95% by weight of paclitaxel,or a pharmaceutically acceptable salt thereof, wherein the particleshave a specific surface area (SSA) of at least 12 m²/g.

Embodiment 39 is the method of embodiment 38, wherein the paclitaxelparticles have a SSA of at least 12 m²/g, 15 m²/g, 20 m²/g, 25 m²/g, 30m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

Embodiment 40 is the method of any one of embodiments 38-39, wherein atleast 40% (w/w) of the paclitaxel is dissolved in 30 minutes or less ina solution of 50% methanol/50% water (v/v) at 37° C. and pH 7.0 in a USPII paddle apparatus operating at 75 RPM.

Embodiment 41 is the method of any one of embodiments 1-22, wherein thechemotherapeutic particles include at least 95% by weight of paclitaxel,wherein at least 40% (w/w) of the paclitaxel is dissolved in 30 minutesor less in a solution of 50% methanol/50% water (v/v) at 37° C. and pH7.0 in a USP II paddle apparatus operating at 75 RPM.

Embodiment 42 is the method of any one of embodiments 1-22, wherein thechemotherapeutic particles include at least 95% by weight of docetaxel,wherein at least 20% (w/w) of the docetaxel is dissolved in 30 minutesor less in a solution of 15% methanol/85% water (v/v) at 37° C. and pH7.0 in a USP II paddle apparatus operating at 75 RPM.

Embodiment 43 is the method of any one of embodiments 1-42, wherein theparticles have a mean particle size number of between about 0.4 μm andabout 1.2 μm, or between about 0.6 μm and about 1.0 μm.

Embodiment 44 is the method of any one of embodiments 1-43, wherein theparticles are uncoated and the composition excludes polymers, proteins,polyethoxylated castor oil, and/or polyethylene glycol glyceridescomposed of mono-, di- and triglycerides and mono- and diesters ofpolyethylene glycol.

Embodiment 45 is the method of any one of embodiments 1-42, wherein thechemotherapeutic particle is present in a suspension further comprisinga pharmaceutically acceptable aqueous carrier.

Embodiment 46 is the method of any one of embodiments 1-45, wherein theparticles comprise at least 96%, 97%, 98%, 99%, or 100% of the compound.

Embodiment 47 is the method of any one of embodiments 1-46, wherein thechemotherapeutic particle is present in a suspension further comprisinga polysorbate, such as polysorbate 80, wherein the polysorbate ispresent in the suspension at a concentration of between about 0.01% v/vand about 1.5% v/v, or between about 0.01% v/v and about 1% v/v, about0.01% v/v and about 0.5% v/v, about 0.01% v/v and about 0.4% v/v, about0.01% v/v and about 0.25% v/v, about 0.05% v/v and about 0.5% v/v, about0.05% v/v and about 0.25% v/v, about 0.1% v/v and about 0.5% v/v, about0.1% v/v and about 0.25% v/v, about 0.1% v/v, about 0.16 v/v, or about0.25% v/v.

Embodiment 48 is the method of embodiment 47, wherein thechemotherapeutic is present in the suspension at a concentration betweenabout 1 mg/ml and about 40 mg/ml, or about 6 mg/ml and about 20 mg/ml.

Embodiment 49 is the method of embodiment 48 wherein thechemotherapeutic particle comprises paclitaxel, or a pharmaceuticallyacceptable salt thereof, wherein the paclitaxel is present in thesuspension at a concentration of between about 1 mg/ml and about 40mg/ml, or about 6 mg/ml and about 20 mg/ml.

Embodiment 50 is the method of embodiment 48 wherein thechemotherapeutic particle comprises docetaxel, or a pharmaceuticallyacceptable salt thereof, wherein the paclitaxel is present in thesuspension at a concentration of between about 1 mg/ml and about 40mg/ml, or about 6 mg/ml and about 20 mg/ml.

Embodiment 51 is a suspension, comprising:

(a) chemotherapeutic particles;

(b) a pharmaceutically acceptable carrier; and

(c) a polysorbate, wherein the polysorbate is present in the suspensionat a concentration of between about 0.01% v/v and about 1.5% v/v, orbetween about 0.01% v/v and about 1% v/v, about 0.01% v/v and about 0.5%v/v, about 0.01% v/v and about 0.4% v/v, about 0.01% v/v and about 0.25%v/v, about 0.05% v/v and about 0.5% v/v, about 0.05% v/v and about 0.25%v/v, about 0.1% v/v and about 0.5% v/v, about 0.1% v/v and about 0.25%v/v, about 0.1% v/v, about 0.16 v/v, or about 0.25% v/v.

Embodiment 52 is a kit, comprising:

(a) a first vial containing chemotherapeutic particles;

(b) a second vial containing a polysorbate and a pharmaceuticallyacceptable carrier;

(c) instructions for reconstituting the chemotherapeutic particles intoa suspension by combining the contents of the first vial and the secondvial, and for diluting the suspension with a diluent solution, such as0.9% saline solution, prior to administration to a patient.

Embodiment 53 is the suspension of embodiment 51 or the kit ofembodiment 52, wherein the chemotherapeutic particles are selected fromthe group consisting of paclitaxel; derivatives of paclitaxel,docetaxel, cabazitaxel, taxanes; epithilones, Vinca alkaloids, such asvinblastine, vincristine, vindesine, vinorelbine; camptothecin analogs;epipodophyllotoxins, such as cisplatin, carboplatin, oxaliplatin,etoposide and teniposide; doxorubicin, anthrcyclines, 5-fluorouracil,topotecan, gemcitabine, peroxisome proliferator-activated receptor(PPAR) ligands, and antiangiogenics, or a pharmaceutically acceptablesalt thereof.

Embodiment 54 is the suspension or kit of any one of embodiments 51-53,wherein the chemotherapeutic is a taxane, or a pharmaceuticallyacceptable salt thereof.

Embodiment 55 is the suspension or kit of any one of embodiments 51-54,wherein the chemotherapeutic is paclitaxel, or a pharmaceuticallyacceptable salt thereof.

Embodiment 56 is the suspension or kit of any one of embodiments 51-54,wherein the chemotherapeutic is docetaxel, or a pharmaceuticallyacceptable salt thereof.

Embodiment 57 is the suspension of any one of embodiments 51 and 53-56,wherein the chemotherapeutic is present in the suspension at aconcentration between about 1 mg/ml and about 40 mg/ml, or about 6 mg/mland about 20 mg/ml.

Embodiment 58 is the suspension or kit of any one of embodiments 51-57,wherein the chemotherapeutic particles comprise at least 95%chemotherapeutic and wherein the particles have a specific surface area(SSA) of at least 10 m²/g, or at least 12 m²/g, 14 m²/g, 16 m²/g, 18m²/g, 20 m²/g, 25 m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

Embodiment 59 is the suspension or kit of any one of embodiments 51-58,wherein the chemotherapeutic particles have an SSA of between about 10m²/g and about 50 m²/g.

Embodiment 60 is the suspension or kit of any one of embodiments 51-59,wherein the chemotherapeutic particles have an SSA of:

(a) between 16 m²/g and 31 m²/g or between 32 m²/g and 50 m²/g;

(b) between 16 m²/g and 30 m²/g or between 32 m²/g and 50 m²/g;

(c) between 16 m²/g and 29 m²/g or between 32 m²/g and 50 m²/g;

(d) between 17 m²/g and 31 m²/g or between 32 m²/g and 50 m²/g;

(e) between 17 m²/g and 30 m²/g or between 32 m²/g and 50 m²/g;

(f) between 17 m²/g and 29 m²/g, or between 32 m²/g and 50 m²/g;

(g) between 16 m²/g and 31 m²/g or between 33 m²/g and 50 m²/g;

(h) between 16 m²/g and 30 m²/g or between 33 m²/g and 50 m²/g;

(i) between 16 m²/g and 29 m²/g or between 33 m²/g and 50 m²/g;

(j) between 17 m²/g and 31 m²/g or between 33 m²/g and 50 m²/g;

(k) between 17 m²/g and 30 m²/g or between 33 m²/g and 50 m²/g;

(l) between 17 m²/g and 29 m²/g, or between 33 m²/g and 50 m²/g;

(m) between 16 m²/g and 31 m²/g, or ≥32 m²/g;

(h) between 17 m²/g and 31 m²/g, or ≥32 m²/g;

(i) between 16 m²/g and 30 m²/g, or ≥32 m²/g;

(j) between 17 m²/g and 30 m²/g, or ≥32 m²/g;

(k) between 16 m²/g and 29 m²/g, or ≥32 m²/g;

(l) between 17 m²/g and 29 m²/g, or ≥32 m²/g;

(m) between 16 m²/g and 31 m²/g, or ≥33 m²/g;

(n) between 17 m²/g and 31 m²/g, or ≥33 m²/g;

(o) between 16 m²/g and 30 m²/g, or ≥33 m²/g;

(p) between 17 m²/g and 30 m²/g, or ≥33 m²/g;

(q) between 16 m²/g and 29 m²/g, or ≥33 m²/g; or

(r) between 17 m²/g and 29 m²/g, or ≥33 m²/g.

Embodiment 61 is the suspension or kit of any one of embodiments 51-60,wherein the chemotherapeutic particles include at least 95% by weight ofa taxane, or a pharmaceutically acceptable salt thereof, wherein theparticles have one or both of the following characteristics:

(i) a mean bulk density between about 0.050 g/cm³ and about 0.15 g/cm³,and/or

(ii) have a specific surface area (SSA) of at least 18 m²/g, 20 m²/g, 25m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

Embodiment 62 is the suspension or kit of any one of embodiments 51-55and 57-61, wherein the taxane is paclitaxel or a pharmaceuticallyacceptable salt thereof, and wherein the particles have a mean bulkdensity between about 0.050 g/cm³ and about 0.12 g/cm³, or between about0.060 g/cm³ and about 0.11 g/cm³.

Embodiment 63 is the suspension or kit of any one of embodiments 51-55and 57-62, wherein the taxane is paclitaxel or a pharmaceuticallyacceptable salt thereof, and wherein the paclitaxel particles have aspecific surface area (SSA) of at least 12 m²/g, 18 m²/g, 20 m²/g, 25m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

Embodiment 64 is the suspension or kit of any one of embodiments 51-55and 57-63, wherein the taxane is paclitaxel or a pharmaceuticallyacceptable salt thereof, and wherein the wherein the paclitaxelparticles have a SSA of between about 22 m²/g and about 40 m²/g, 25 m²/gand about 40 m²/g, 30 m²/g and about 40 m²/g, or between about 35 m²/gand about 40 m²/g.

Embodiment 65 is the suspension or kit of any one of embodiments 51-55and 57-64, wherein the paclitaxel particles have a bulk density ofbetween about 0.060 g/cm³ and about 0.11 g/cm³ and a SSA of betweenabout 22 m²/g and about 40 m²/g.

Embodiment 66 is the suspension or kit of any one of embodiments 51-55and 57-65, wherein at least 40% (w/w) of the paclitaxel is dissolved in30 minutes or less in a solution of 50% methanol/50% water (v/v) at 37°C. and pH 7.0 in a USP II paddle apparatus operating at 75 RPM.

Embodiment 67 is the suspension or kit of any one of embodiments 51-54and 56-61, wherein the taxane is docetaxel or a pharmaceuticallyacceptable salt thereof, and wherein the particles have a mean bulkdensity between about 0.050 g/cm³ and about 0.12 g/cm³, or between about0.06 g/cm³ and about 0.1 g/cm³.

Embodiment 68 is the suspension or kit of any one of embodiments 51-54,56-61, and 67 wherein the taxane is docetaxel or a pharmaceuticallyacceptable salt thereof, and wherein the docetaxel particles have a SSAof at least 18 m²/g, 20 m²/g, 25 m²/g, 30 m²/g, 35 m²/g, 40 m²/g, or 42m²/g.

Embodiment 69 is the suspension or kit of any one of embodiments 51-54,56-61, and 67-68, wherein the taxane is docetaxel or a pharmaceuticallyacceptable salt thereof, and wherein the docetaxel particles have a SSAof between about 40 m²/g and about 50 m²/g, or between about 43 m²/g andabout 46 m²/g.

Embodiment 70 is the suspension or kit of any one of embodiments 51-54,56-61, and 67-69, wherein the docetaxel particles have a bulk density ofbetween about 0.06 g/cm³ and about 0.1 g/cm³ and a SSA of between about40 m²/g and about 50 m²/g.

Embodiment 71 is the suspension or kit of any one of embodiments 51-54,56-61, and 67-70, wherein at least 20% (w/w) of the docetaxel isdissolved in 30 minutes or less in a solution of 15% methanol/85% water(v/v) at 37° C. and pH 7.0 in a USP II paddle apparatus operating at 75RPM.

Embodiment 72 is the suspension or kit of any one of embodiments 51-71,wherein the particles have a mean particle size number of between about0.4 μm and about 1.2 μm, or between about 0.6 μm and about 1.0 μm.

Embodiment 73 is the suspension or kit of any one of embodiments 51-72,wherein the particles are uncoated and the suspension or kit excludespolymers, proteins, polyethoxylated castor oil, and/or polyethyleneglycol glycerides composed of mono-, di- and triglycerides and mono- anddiesters of polyethylene glycol.

Embodiment 74 is the suspension or kit of any one of embodiments 51-73,wherein the pharmaceutically acceptable carrier is saline, such as 0.9%sodium chloride solution.

Embodiment 75 is the suspension or kit of any one of embodiments 51-74,wherein the polysorbate is polysorbate 80.

Embodiment 76 is the kit of any one of embodiments 52-75, wherein thecontents of the first and second vial are sterile.

Embodiment 77 is the method, suspension, or kit of any one ofembodiments 1-76, wherein the chemotherapeutic particles arenon-agglomerated individual particles.

DESCRIPTION OF THE FIGURES

FIG. 1. Median tumor volume of PC3 human prostate carcinoma in micetreated by direct tumor injection (ITU) with vehicle and paclitaxelparticles (Study P-PPr-01-2015).

FIG. 2. Median tumor volume for mouse PC-3 prostate tumor xenograftstreated IT with vehicle, NanoDoce™, NanoPac™ and treated IV withdocetaxel (Study PD-PPr-02-2016).

FIG. 3. Mean tumor volume for mouse MDA-MB-231 xenografts treated ITwith vehicle, NanoDoce™, NanoPac™ and treated IV with docetaxel orpaclitaxel (Study PD-PB-04-2016).

FIG. 4. Mean tumor volume for mouse MX-1 xenografts treated IT withvehicle, NanoDoce™, NanoPac™ and treated IV with docetaxel or paclitaxel(Study P-PB-03-2016).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used herein, “about” means+/−five percent (5%) of the recited unit ofmeasure.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication. The compositions and methods for their use can “comprise,”“consist essentially of,” or “consist of” any of the ingredients orsteps disclosed throughout the specification. With respect to the phrase“consisting essentially of,” a basic and novel property of thecompositions of the present invention are their ability to treat solidtumors by direct injection of uncoated (neat) chemotherapeuticparticles. This can be achieved without the use of coatings,encapsulations, and other drug delivery aids.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While the specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

In one aspect, the invention provides methods for treating a solidtumor, comprising administering to a subject with a solid tumor anamount effective of a composition comprising chemotherapeutic particlesto treat the tumor, wherein the composition is directly injected intothe tumor. The inventors have surprisingly discovered thatchemotherapeutic particles administered according to the methods of theinvention can accumulate in very high levels in the tumor for anextended period of time with little leakage of the chemotherapeuticparticles to undesired locations. As will be understood by those ofskill in the art, free drug administered directly into tumors isinadequately retained in the tumor for entry into tumor cells foroptimal therapeutic benefit. Thus, the methods of the invention providea significant improvement over prior art methods. The chemotherapeuticparticles exhibit a much higher surface area compared tochemotherapeutic particles prepared by typical procedures. This allowsfor the particles injected into the tumor to be too large to be carriedaway by systemic circulation and yet release the chemotherapeutic agentmuch faster than traditional particles.

As used herein, “chemotherapeutic particles” are particles consistingessentially of the chemotherapeutic (i.e.: at least 95%, 96%, 97%, 98%,99%, or 100% chemotherapeutic) that are between 0.1 μm and 5 μm indiameter. Chemotherapeutic particles are different than “particlescontaining chemotherapeutic”, which are particles that containchemotherapeutic and at least one added excipient. Chemotherapeuticparticles of the invention are uncoated, and are not embedded,contained, enclosed or encapsulated within a solid excipient.Chemotherapeutic particles of the invention may, however, containimpurities and byproducts typically found during preparation of thechemotherapeutic. Even so, chemotherapeutic particles comprise at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%chemotherapeutic, meaning the chemotherapeutic particles consist of orconsist essentially of substantially pure chemotherapeutic.

In other embodiments, the chemotherapeutic particles are greater than0.2 μm, or 0.3 μm in diameter. In another embodiment, thechemotherapeutic particles are at least 0.4 μm diameter. In furtherembodiments, the chemotherapeutic particles are between 0.4 μm and 2 μmin diameter, or between 0.5 μm and 1.5 μm in diameter, or between 0.2 μmand 1 μm in diameter, or between 0.2 μm to less than 1 μm in diameter.In further embodiments, the chemotherapeutic particles can have a meanparticle size number of between in the range of about 0.4 μm to about 5μm, about 0.4 μm to about 3 μm, about 0.5 μm to about 1.4 μm, about 0.4μm to about 0.8 μm, about 0.4 μm to about 0.7 μm, or about 0.5 μm toabout 0.7 μm. In a further embodiment, the chemotherapeutic orpaclitaxel particles have a mean particle size number of between about0.4 μm and about 1.2 μm, or between about 0.6 μm and about 1.0 μm. Inanother embodiment, the chemotherapeutic or paclitaxel particles have amean particle size number of between 0.6 μm and 0.861 μm, or betweenabout 0.5 μm to about 0.7 μm, or between about 0.2 μm to about 1 μm, orbetween about 0.2 μm to less than 1 μm, or between about 0.3 μm to about1 μm, or between about 0.3 μm to less than 1 μm, or between about 0.4 μmto about 1 μm, or between about 0.4 μm to less than 1 μm.

Various processes are disclosed in U.S. Pat. Nos. 5,833,891 6,113,795,8,221,779, and WO2016/197091, which are incorporated by reference hereinin their entireties, for producing particle sizes as small as 0.1 to 5μm for compounds.

As used herein, a “solid tumor” is an abnormal mass of tissue thatusually does not contain cysts or liquid areas. Solid tumors may bebenign (not cancer) or malignant (cancer). Different types of solidtumors are named for the type of cells that form them. Examples of solidtumors are sarcomas, carcinomas, and lymphomas. In one particularembodiment, the solid tumor is a malignant solid tumor.

As used herein, “directly injected into the tumor” means that some orall of the composition, such as a suspension, is injected into the tumormass. As will be understood by those of skill in the art, such directinjection may include injection of some portion of the composition, suchas a suspension, for example, drug on the periphery of the solid tumor(“peritumorally”), such as if the amount of composition or suspensionthereof is too large to all be directly injected into the solid tumormass. In one embodiment, the composition or suspension thereof isinjected in its entirety into the solid tumor mass. As used herein thetumor includes both the tumor mass and tumor metastases, including butnot limited to bone and soft tissue metastases.

As used herein, “treat” or “treating” means accomplishing one or more ofthe following: (a) reducing tumor size; (b) reducing tumor growth; (c)reducing or limiting development and/or spreading of metastases; (d)reducing or limiting development of one or more side effects ofchemotherapy treatment.

Side effects of chemotherapy treatment include, but are not limited toanemia, neutropenia, thrombocytopenia, neurologic toxicities, reductionin appetite, constipation, diarrhea, hair loss, fatigue,nausea/vomiting, and pain.

In one embodiment, treating the tumor comprises inhibiting tumormetastasis. “Inhibiting” tumor cell metastasis may comprise any amountof inhibition compared to no treatment. In various non-limitingembodiments, the methods may comprise inhibiting development of tumorcell metastasis, or reducing existing tumor metastases by 5%, 10%, 25%,50%; 100%, or more compared to control (such as no treatment).

In another aspect, the invention provides methods for inhibiting tumormetastasis, comprising administering to a subject with a tumor an amounteffective of a composition comprising chemotherapeutic particles toinhibit tumor metastasis.

The inventors have surprisingly discovered that chemotherapeutic agentsdescribed herein administered to a subject migrate into, and areretained in, the lymphatic system of the subject. As will be understoodby those of skill in the art, tumors are profused with circulatingblood, and free drug administered directly into tumors is inadequatelyretained in the tumor for entry into tumor cells for optimal therapeuticbenefit. Malignant cells within a tumor spread by way of the bloodsystem, the lymphatic system to lymph nodes, by migration of cancercells within the fluids of the peritoneal cavity, and to distant sitesin a process known as metastasis. Thus, the methods of the invention canbe used to inhibit metastasis. In this aspect, the chemotherapeuticparticles can be administered via direct injection, intraperitonealinjection, peritumoral injection, or other suitable administrativeroute.

“Inhibiting” tumor cell metastasis may comprise any amount of inhibitioncompared to no treatment. In various non-limiting embodiments, themethods may comprise inhibiting development of tumor cell metastasis, orreducing existing tumor metastases, by 5%, 10%, 25%, 50%; 100%, or morecompared to control (such as no treatment).

In various embodiments, the chemotherapeutic is selected from the groupconsisting of taxanes (paclitaxel, derivatives of paclitaxel, docetaxel,cabazitaxel, etc.), epithilones, Vinca alkaloids, such as vinblastine,vincristine, vindesine, vinorelbine; camptothecin analogs;epipodophyllotoxins, such as cisplatin, carboplatin, oxaliplatin,etoposide and teniposide; doxorubicin, anthrcyclines, 5-fluorouracil,topotecan, gemcitabine, peroxisome proliferator-activated receptor(PPAR) ligands, and antiangiogenics.

In various embodiments, the solid tumor is selected from the groupconsisting of sarcomas, carcinomas, and lymphomas, breast tumors,prostate tumors, head and neck tumors, glioblastomas, bladder tumors,pancreatic tumors, liver tumors, ovarian tumors, colorectal tumors,pulmonary, cutaneous, lymphoid, and gastrointestinal tumors. In aspecific embodiment, the solid tumor is a prostate tumor and thechemotherapeutic particles are paclitaxel or docetaxel particles. Inanother specific embodiment, the solid tumor is an ovarian tumor and thechemotherapeutic particles are paclitaxel or docetaxel particles. Inanother specific embodiment, the solid tumor is a breast tumor and thechemotherapeutic particles are docetaxel particles. In another specificembodiment, the solid tumor is a pancreatic tumor and thechemotherapeutic particles are paclitaxel or docetaxel particles. In anyof these embodiments, the tumor may be, for example, an adenocarcinoma.

The inventors have unexpectedly been able to produce chemotherapeuticparticles, such as taxane particles, that have a mean bulk densitybetween about 0.050 g/cm³ and about 0.15 g/cm³, and/or a specificsurface area (SSA) of at least 18 m²/g an SSA using novel methods forproducing the particles as described below. As shown in the examplesthat follow, the increased specific surface area and decreased bulkdensity of the taxane particles result in significant increases indissolution rate compared to the raw taxane and to milled taxaneproducts used for comparison. Dissolution takes place only at asolid/liquid interface. Therefore, increased specific surface area willincrease the dissolution rate due to a larger number of molecules on thesurface of the particle having contact with the dissolution media. Thebulk density takes into account the macrostructure and inter-particlespace of a powder. Parameters that contribute to the bulk densityinclude particle size distribution, particle shape, and the affinity ofthe particles for each other (i.e., agglomeration). Lower powder bulkdensities yield faster dissolution rates. This is due to the ability ofthe dissolution media to more readily penetrate the interstitial orinter-particle spaces and have greater contact with the surface of theparticles. Therefore, each of the increased specific surface area andthe decreased bulk density result in the significant increase indissolution rate for the taxane particles of the invention compared tothe unprocessed or raw material, and the milled taxane product used forcomparison. This provides a significant improvement for use of thetaxane particles of the invention in, for example, tumor treatment.

Thus, in another embodiment, the chemotherapeutic particles have aspecific surface area (SSA) of at least 10 m²/g, or at least 12 m²/g, 14m²/g, 16 m²/g, 18 m²/g, 20 m²/g, 25 m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or35 m²/g. In one embodiment, the chemotherapeutic particles have an SSAof between about 10 m²/g and about 50 m²/g.

In these embodiments, it is preferred that the chemotherapeuticparticles are taxane particles. Taxanes are a class of diterpenoidscontaining a taxadiene core that are very poorly soluble in water. Thetaxane particles of the invention may be any suitable taxane, includingbut not limited to paclitaxel, docetaxel, cabazitaxel, taxadiene,baccatin III, taxchinin A, brevifoliol, and taxuspine D, combinationsthereof, or pharmaceutically acceptable salts thereof. In oneembodiment, the taxane is selected from the group consisting ofpaclitaxel, docetaxel, and cabazitaxel, or a pharmaceutically acceptablesalt thereof. In various embodiments of the present invention, thetaxane particles are uncoated (neat) individual particles; the taxaneparticles are not bound to or conjugated to any substance; no substancesare absorbed or adsorbed onto the surface of the taxane particles; thetaxane particles are not encapsulated in any substance; the taxaneparticles are not coated with any substance; the taxane particles arenot microemulsions, nanoemulsions, microspheres, or liposomes of ataxane; and/or the taxane particles are not bound to, attached to,encapsulated in, or coated with a monomer, a polymer (or biocompatiblepolymer), a protein, a surfactant, or albumin. In some embodiments, amonomer, a polymer (or biocompatible polymer), a copolymer, a protein, asurfactant, or albumin is not absorbed or adsorbed onto the surface ofthe taxane particles. In some embodiments, the compositions are freeof/do not include or contain a polymer/copolymer or biocompatiblepolymer/copolymer. In some embodiments, the compositions are free of/donot include or contain a protein. In some aspects of the invention, thecompositions are free of/do not include or contain albumin. In someaspects of the invention, the compositions are free of/do not include orcontain hyaluronic acid. In some aspects of the invention, thecompositions are free of/do not include or contain a conjugate ofhyaluronic acid and a taxane. In some aspects of the invention, thecompositions are free of/do not include or contain a conjugate ofhyaluronic acid and paclitaxel. In some aspects of the invention, thecompositions are free of/do not include or contain poloxamers,polyanions, polycations, modified polyanions, modified polycations,chitosan, chitosan derivatives, metal ions, nanovectors,poly-gamma-glutamic acid (PGA), polyacrylic acid (PAA), alginic acid(ALG), Vitamin E-TPGS, dimethyl isosorbide (DMI), methoxy PEG 350,citric acid, anti-VEGF antibody, ethylcellulose, polystyrene,polyanhydrides, polyhydroxy acids, polyphosphazenes, polyorthoesters,polyesters, polyamides, polysaccharides, polyproteins,styrene-isobutylene-styrene (SIBS), a polyanhydride copolymer,polycaprolactone, polyethylene glycol (PEG), Poly(bis(P-carboxyphenoxy)propane-sebacic acid, poly(d,l-lactic acid) (PLA),poly(d.l-lactic acid-co-glycolic acid) (PLAGA), and/or poly(D, Llactic-co-glycolic acid (PLGA).

In one such embodiment, the chemotherapeutic particles include at least95% by weight of a taxane, or a pharmaceutically acceptable saltthereof, wherein the particles have one or both of the followingcharacteristics:

(i) a mean bulk density between about 0.050 g/cm³ and about 0.15 g/cm³,and/or

(ii) have a specific surface area (SSA) of at least 18 m²/g, 20 m²/g, 25m²/g, 30 m²/g, 32 m²/g, 34 m²/g, or 35 m²/g.

As used herein, the “specific surface area” is the total surface area ofthe taxane particle per unit of taxane mass as measured by theBrunauer-Emmett-Teller (“BET”) isotherm (i.e.: the BET SSA). As will beunderstood by those of skill in the art, the “taxane particles” caninclude both agglomerated taxane particles and non-agglomerated taxaneparticles; since the SSA is determined on a per gram basis it takes intoaccount both agglomerated and non-agglomerated taxane particles in thecomposition. The BET specific surface area test procedure is acompendial method included in both the United States Pharmaceopeia andthe European Pharmaceopeia.

As used herein, the bulk density of the taxane particles is the mass ofthe totality of particles in the composition divided by the total volumethey occupy when poured into a graduated cylinder, without tapping thegraduated cylinder. The total volume includes particle volume,inter-particle void volume, and internal pore volume.

In one embodiment, the taxane is paclitaxel or a pharmaceuticallyacceptable salt thereof, and the particles have a mean bulk densitybetween about 0.050 g/cm³ and about 0.12 g/cm³. In another embodiment,the paclitaxel particles have a mean bulk density between about 0.060g/cm³ and about 0.11 g/cm³. In some embodiments, the chemotherapeuticand taxane particles are non-agglomerated individual particles and arenot clusters of multiple chemotherapeutic particles.

In a further embodiment, the taxane is paclitaxel or a pharmaceuticallyacceptable salt thereof, and the paclitaxel particles have a specificsurface area (SSA) of at least 12 m²/g. In various embodiments, thepaclitaxel particles have an SSA of at least 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or at least 40 m²/g. In various further embodiments, thepaclitaxel particles have an SSA of between about 12 m²/g and about 40m²/g, between about 14 m²/g and about 40 m²/g, between about 15 m²/g andabout 40 m²/g, between about 16 m²/g and about 40 m²/g, between about 17m²/g and about 40 m²/g, between about 18 m²/g and about 40 m²/g, betweenabout 19 m²/g and about 40 m²/g, between about 20 m²/g and about 40m²/g, between about 22 m²/g and about 40 m²/g, between about 25 m²/g andabout 40 m²/g, between about 26 m²/g and about 40 m²/g, between about 30m²/g and about 40 m²/g, between about 35 m²/g and about 40 m²/g, betweenabout 20 m²/g and about 29 m²/g, between about 20 m²/g and about 28m²/g, between about 20 m²/g and about 26.2 m²/g, between about 22 m²/gand about 29 m²/g, between about 22 m²/g and about 28 m²/g, betweenabout 22 m²/g and about 26.2 m²/g, between about 32 m²/g and about 40m²/g, between about 32 m²/g and about 38 m²/g, or between about 32 m²/gand about 36 m²/g.

In one preferred embodiment, the paclitaxel particles have a mean bulkdensity of between about between about 0.050 g/cm³ and about 0.12 g/cm³and a SSA of at least 30 m²/g. In another preferred embodiment, thepaclitaxel particles have a mean bulk density of between about betweenabout 0.050 g/cm³ and about 0.12 g/cm³ and a SSA of at least 35 m²/g. Inone the paclitaxel particles have a mean bulk density of between aboutbetween about 0.050 g/cm³ and about 0.12 g/cm³ and a SSA of betweenabout 30 m²/g and about 40 m²/g. In another preferred embodiment, thepaclitaxel particles have a mean bulk density of between about 0.060g/cm³ and about 0.11 g/cm³ and a SSA of between about 30 m²/g and about40 m²/g. In another preferred embodiment, the paclitaxel particles havea mean bulk density of between about 0.060 g/cm³ and about 0.11 g/cm³and a SSA of at least 30 m²/g. In a further embodiment, the paclitaxelparticles have a mean bulk density of between about 0.060 g/cm³ andabout 0.11 g/cm³ and a SSA of at least 35 m²/g. These variousembodiments are exemplified in the examples that follow.

In any of these various embodiments, the paclitaxel particles mayinclude at least 4.16×10⁻¹³ gram paclitaxel, or a pharmaceuticallyacceptable salt thereof per paclitaxel particle.

In another embodiment, at least 40% (w/w) of the paclitaxel in thepaclitaxel particles of the composition is dissolved in 30 minutes orless in a solution of 50% methanol/50% water (v/v) in a USP II paddleapparatus operating at 75 RPM. pH 7 was used, and the solubility of thetaxanes are not effected by pH. In another embodiment, the dissolutionstudies are carried out at 37° C.

In another embodiment, the taxane is docetaxel or a pharmaceuticallyacceptable salt thereof, and the docetaxel particles have a mean bulkdensity between about 0.050 g/cm³ and about 0.12 g/cm³. In a furtherembodiment, the mean bulk density of the docetaxel particles is betweenabout 0.06 g/cm³ and about 0.1 g/cm³.

In another embodiment, the taxane is docetaxel or a pharmaceuticallyacceptable salt thereof, and wherein the docetaxel particles have a SSAof at least 18 m²/g. In various further embodiments, the docetaxelparticles have a SSA of at least 20 m²/g, 25 m²/g, 30 m²/g, 35 m²/g, 40m²/g, or 42 m²/g. In a further embodiment, the docetaxel particles havea SSA of between about 40 m²/g and about 50 m²/g. In another embodiment,the docetaxel particles have a SSA of between about 43 m²/g and about 46m²/g.

In one preferred embodiment, the docetaxel particles have a mean bulkdensity between about 0.050 g/cm³ and about 0.12 g/cm³ and a SSA of atleast 30 m²/g. In another preferred embodiment, the docetaxel particleshave a mean bulk density between about 0.050 g/cm³ and about 0.12 g/cm³and a SSA of at least 35 m²/g. In a further preferred embodiment, thedocetaxel particles have a mean bulk density between about 0.050 g/cm³and about 0.12 g/cm³ and a SSA of at least 40 m²/g. In one preferredembodiment, the docetaxel particles have a mean bulk density betweenabout 0.050 g/cm³ and about 0.12 g/cm³ and a SSA of between about 40m²/g and about 50 m²/g. In another preferred embodiment, mean bulkdensity of the docetaxel particles is between about 0.06 g/cm³ and about0.1 g/cm³ and the SSA is between about 40 m²/g and about 50 m²/g. Thesevarious embodiments are exemplified in the examples that follow.

In any of these various embodiments, the docetaxel particles may includeat least 4.16×10⁻¹³ grams docetaxel, or a pharmaceutically acceptablesalt thereof per docetaxel particle.

In another embodiment, at least 20% (w/w) of the docetaxel is dissolvedin 30 minutes or less in a solution of 15% methanol/85% water (v/v) in aUSP II paddle apparatus operating at 75 RPM. A neutral pH was used wherethe solubility of the taxanes are not effected by pH. In anotherembodiment, the dissolution studies are carried out at 37° C. In afurther embodiment, the chemotherapeutic particles include at least 95%by weight of paclitaxel, or a pharmaceutically acceptable salt thereof,wherein the particles have a specific surface area (SSA) of:

(a) between 16 m²/g and 31 m²/g or between 32 m²/g and 40 m²/g;

(b) between 16 m²/g and 30 m²/g or between 32 m²/g and 40 m²/g;

(c) between 16 m²/g and 29 m²/g or between 32 m²/g and 40 m²/g;

(d) between 17 m²/g and 31 m²/g or between 32 m²/g and 40 m²/g;

(e) between 17 m²/g and 30 m²/g or between 32 m²/g and 40 m²/g;

(f) between 17 m²/g and 29 m²/g, or between 32 m²/g and 40 m²/g;

(g) between 16 m²/g and 31 m²/g or between 33 m²/g and 40 m²/g;

(h) between 16 m²/g and 30 m²/g or between 33 m²/g and 40 m²/g;

(i) between 16 m²/g and 29 m²/g or between 33 m²/g and 40 m²/g;

(j) between 17 m²/g and 31 m²/g or between 33 m²/g and 40 m²/g;

(k) between 17 m²/g and 30 m²/g or between 33 m²/g and 40 m²/g;

(l) between 17 m²/g and 29 m²/g, or between 33 m²/g and 40 m²/g;

(m) between 16 m²/g and 31 m²/g, or ≥32 m²/g;

(h) between 17 m²/g and 31 m²/g, or ≥32 m²/g;

(i) between 16 m²/g and 30 m²/g, or ≥32 m²/g;

-   -   (j) between 17 m²/g and 30 m²/g, or ≥32 m²/g;    -   (k) between 16 m²/g and 29 m²/g, or ≥32 m²/g;    -   (1) between 17 m²/g and 29 m²/g, or ≥32 m²/g;

(m) between 16 m²/g and 31 m²/g, or ≥33 m²/g;

(n) between 17 m²/g and 31 m²/g, or ≥33 m²/g;

(o) between 16 m²/g and 30 m²/g, or ≥33 m²/g;

(p) between 17 m²/g and 30 m²/g, or ≥33 m²/g;

(q) between 16 m²/g and 29 m²/g, or ≥33 m²/g; or

(r) between 17 m²/g and 29 m²/g, or ≥33 m²/g.

In another embodiment, at least 40% (w/w) of the paclitaxel, or apharmaceutically acceptable salt thereof, in the paclitaxel particles ofthe composition is dissolved in 30 minutes or less in a solution of 50%methanol/50% water (v/v) in a USP II paddle apparatus operating at 75RPM. pH 7 was used, and the solubility of the taxanes are not effectedby pH. In another embodiment, the dissolution studies are carried out at37° C.

In a further embodiment, the chemotherapeutic particles include at least95% by weight of paclitaxel, or a pharmaceutically acceptable saltthereof, wherein at least 40% (w/w) of the paclitaxel is dissolved in 30minutes or less in a solution of 50% methanol/50% water (v/v) in a USPII paddle apparatus operating at 75 RPM. pH 7 was used, and thesolubility of the taxanes are not effected by pH. In another embodiment,the dissolution studies are carried out at 37° C.

In a further embodiment, the chemotherapeutic particles include at least95% by weight of docetaxel, or a pharmaceutically acceptable saltthereof, wherein at least 20% (w/w) of the docetaxel is dissolved in 30minutes or less in a solution of 15% methanol/85% water (v/v) in a USPII paddle apparatus operating at 75 RPM. pH 7 was used, and thesolubility of the taxanes are not effected by pH. In another embodiment,the dissolution studies are carried out at 37° C.

In one embodiment, the chemotherapeutic particles comprises a dosageform of chemotherapeutic in suspension (i.e.: with a pharmaceuticallyacceptable carrier and any other components) of between about 0.1 mg/mland about 100 mg/ml chemotherapeutic. In various further embodiments,the dosage form may be between about 0.5 mg/ml and about 100 mg/ml,about 1 mg/ml and about 100 mg/ml, about 2 mg/ml and about 100 mg/ml,about 5 mg/ml and about 100 mg/ml, about 10 mg/ml and about 100 mg/ml,about 25 mg/ml and about 100 mg/ml, about 0.1 mg/ml and about 75 mg/ml,about 0.5 mg/ml and about 75 mg/ml, about 1 mg/ml and about 75 mg/ml,about 2 mg/ml and about 75 mg/ml, about 5 mg/ml and about 75 mg/ml,about 10 mg/ml and about 75 mg/ml, about 25 mg/ml and about 75 mg/m,about 0.1 mg/ml and about 50 mg/ml, about 0.5 mg/ml and about 50 mg/ml,about 1 mg/ml and about 50 mg/ml, about 2 mg/ml and about 50 mg/ml,about 5 mg/ml and about 50 mg/ml, about 10 mg/ml and about 50 mg/ml,about 25 mg/ml and about 50 mg/m, about 0.1 mg/ml and about 25 mg/ml,about 0.5 mg/ml and about 25 mg/ml, about 1 mg/ml and about 40 mg/ml,about 1 mg/ml and about 25 mg/ml, about 2 mg/ml and about 25 mg/ml,about 5 mg/ml and about 25 mg/ml, about 10 mg/ml and about 25 mg/ml,about 0.1 mg/ml and about 15 mg/ml, about 0.5 mg/ml and about 15 mg/ml,about 1 mg/ml and about 15 mg/ml, about 2 mg/ml and about 15 mg/ml,about 5 mg/ml and about 15 mg/ml, about 10 mg/ml and about 15 mg/ml,about 0.1 mg/ml and about 10 mg/ml, about 0.5 mg/ml and about 10 mg/ml,about 1 mg/ml and about 10 mg/ml, about 2 mg/ml and about 10 mg/ml,about 5 mg/ml and about 10 mg/ml, about 0.1 mg/ml and about 5 mg/ml,about 0.5 mg/ml and about 5 mg/ml, about 1 mg/ml and about 5 mg/ml,about 2 mg/ml and about 5 mg/ml, about 0.1 mg/ml and about 2 mg/ml,about 0.5 mg/ml and about 2 mg/ml, about 1 mg/ml and about 2 mg/ml,about 0.1 mg/ml and about 1 mg/ml, about 0.5 mg/ml and about 1 mg/ml,about 0.1 mg/ml and about 0.5 mg/ml, about 0.1 mg/ml and about 15 mg/ml,about 0.5 mg/ml and about 15 mg/ml, about 1 mg/ml and about 15 mg/ml,about 2 mg/ml and about 15 mg/ml, about 5 mg/ml and about 15 mg/ml,about 3 mg/ml and about 8 mg/ml, or about 4 mg/ml and about 6 mg/mlpaclitaxel, or at least about 0.1, 0.5, 1, 10, 20, 25, 50, 75, or 100mg/ml chemotherapeutic.

In all aspects and embodiments of the invention, the composition may beprovided in a suitable pharmaceutically acceptable carrier as deemedappropriate by attending medical personnel. In one embodiment, theformulation comprises a small volume (i.e.: 10 ul-40 ml; in otherembodiments, 10 ul-35 ml, 10 ul-30 ml, 10 ul-25 ml, 10 ul-20 ml, 10ul-15 ml, 10 ul-10 ml, 10 ul-7.5 ml, 10 ul-5 ml, 10 ul-4 ml, 10 ul-3 ml,50 ul-40 ml, 50 ul-35 ml, 50 ul-30 ml, 50 ul-25 ml, 50 ul-20 ml, 50ul-15 ml, 50 ul-10 ml, 50 ul-7.5 ml 50 ul-5 ml, 50 ul-4 ml, 50 ul-3 ml,100 ul-40 ml, 100 ul-35 ml, 100 ul-30 ml, 100 ul-25 ml, 100 ul-20 ml,100 ul-15 ml, 100 ul-10 ml, 100 ul-7.5 ml 100 ul-5 ml, 100 ul-5 ml, 100ul-4 ml, 100 ul-3 ml, 500 ul-40 ml, 500 ul-35 ml, 500 ul-30 ml, 500ul-25 ml, 500 ul-20 ml, 500 ul-15 ml, 500 ul-10 ml, 500 ul-7.5 ml, 500ul-5 ml, 500 ul-4 ml, 500 ul-3 ml, 1 ml-40 ml, 1 ml-35 ml, 1 ml-30 ml, 1ml-25 ml, 1 ml-20 ml, 1 ml-15 ml, 1 ml-10 ml, 1 ml-7.5 ml 1 ml-5 ml, 1ml-4 ml, or 1 ml-3 ml). Thus, in another aspect, the invention providespharmaceutical compositions comprising a chemotherapeutic particle and apharmaceutically acceptable carrier, where the total volume of thecomposition is between 1 ml and 40 ml, 1 ml and 35 ml, 1 ml and 30 ml, 1ml and 25 ml, 1 ml and 20 ml, 1 ml and 15 ml, 1 ml and 10 ml, 1 ml and7.5 ml, 1 ml and 5 ml, 1 ml and 4 ml, or 1 ml and 3 ml. In oneembodiment, the composition consists of the chemotherapeutic particlesand a pharmaceutically acceptable carrier, such as a liquid, semi-solid,or solid carrier.

In one embodiment, the composition comprises or consists of thechemotherapeutic particles and a liquid carrier. Any suitable liquidcarrier may be used, such as an aqueous liquid carrier. Any suitableaqueous liquid carrier can be used, including but not limited to 0.9%saline solution (normal saline) such as 0.9% Sodium Chloride forInjection USP. In another embodiment, the composition comprises asuspension. In some embodiments, the suspension includes an aqueouscarrier. The carrier can comprise buffering agent, osmotic salt and/orsurfactant in water, and other agents for adjustment of pH, isotonicityand viscosity. In one embodiment employing an aqueous carrier, theconcentration of surfactant is less than 1% on a w/w or w/v basis; inother embodiments, less than 0.5%, less than 0.25%, or about 0.1%. Inother embodiments, the aqueous carrier can exclude the surfactantsGELUCIRE® (polyethylene glycol glycerides composed of mono-, di- andtriglycerides and mono- and diesters of polyethylene glycol) and/orCREMOPHOR® (polyethoxylated castor oil). In some embodiments, thecomposition or suspension excludes polymers, proteins (such as albumin),polyethoxylated castor oil, and/or polyethylene glycol glyceridescomposed of mono-, di- and triglycerides and mono- and diesters ofpolyethylene glycol.

The suspension can be administered as is or can be diluted with adiluent, e.g. with saline water (0.9% saline solution) optionallyincluding a buffering agent and one or more other excipients, prior toadministration. The diluent can be any liquid suitable foradministration to a patient by injection. For example, the volume ratioof suspension to diluent might be in the range of 1:1-1:100 v/v or othersuitable ratio.

In some embodiments, the suspension can comprise water and optionallyone or more excipients selected from the group consisting of buffer,tonicity adjusting agent, preservative, demulcent, viscosity modifier,osmotic agent, surfactant, antioxidant, alkalinizing agent, acidifyingagent, antifoaming agent, and colorant. For example, the suspension cancomprise chemotherapeutic particles, water, buffer and salt. Itoptionally further comprises a surfactant. In some embodiments, thesuspension consists essentially of or consists of water, paclitaxelparticles suspended in the water and buffer. The suspension can furthercontain an osmotic salt.

The suspension can comprise one or more surfactants. Suitablesurfactants include by way of example and without limitationpolysorbates, lauryl sulfates, acetylated monoglycerides, diacetylatedmonoglycerides, and poloxamers. Polysorbates are polyoxyethylenesorbitan fatty acid esters which are a series of partial fatty acidesters of sorbitol and its anhydrides copolymerized with approximately20, 5, or 4 moles of ethylene oxide for each mole of sorbitol and itsanhydrides. Non-limiting examples of polysorbates are polysorbate 20,polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61,polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, andpolysorbate 120. Polysorbates containing approximately 20 moles ofethylene oxide are hydrophilic nonionic surfactants. Examples ofpolysorbates containing approximately 20 moles of ethylene oxide includepolysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,polysorbate 80, polysorbate 85, and polysorbate 120. Polysorbates areavailable commercially from Croda under the tradename TWEEN™. The numberdesignation of the polysorbate corresponds to the number designation ofthe TWEEN, e.g., polysorbate 20 is TWEEN 20, polysorbate 40 is TWEEN 40,polysorbate 60 is TWEEN 60, polysorbate 80 is TWEEN 80, etc. USP/NFgrades of polysorbate include polysorbate 20 NF, polysorbate 40 NF,polysorbate 60 NF, and polysorbate 80 NF. Polysorbates are alsoavailable in PhEur grades (European Pharmacopoeia), BP grades, and JPgrades. The term “polysorbate” is a non-proprietary name. The chemicalname of polysorbate 20 is polyoxyethylene 20 sorbitan monolaurate. Thechemical name of polysorbate 40 is polyoxyethylene 20 sorbitanmonopalmitate. The chemical name of polysorbate 60 is polyoxyethylene 20sorbitan monostearate. The chemical name of polysorbate 80 ispolyoxyethylene 20 sorbitan monooleate. In some embodiments, thesuspension can comprise mixtures of polysorbates. In some embodiments,the suspension comprises polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 65, polysorbate 80, polysorbate 85, and/or polysorbate 120.In other embodiments, the suspension comprises polysorbate 20,polysorbate 40, polysorbate 60, and/or polysorbate 80. In oneembodiment, the suspension comprises polysorbate 80.

The suspension can comprise one or more tonicity adjusting agents.Suitable tonicity adjusting agents include by way of example and withoutlimitation, one or more inorganic salts, electrolytes, sodium chloride,potassium chloride, sodium phosphate, potassium phosphate, sodium,potassium sulfates, sodium and potassium bicarbonates and alkaline earthmetal salts, such as alkaline earth metal inorganic salts, e.g., calciumsalts, and magnesium salts, mannitol, dextrose, glycerin, propyleneglycol, and mixtures thereof.

The suspension can comprise one or more buffering agents. Suitablebuffering agents include by way of example and without limitation,dibasic sodium phosphate, monobasic sodium phosphate, citric acid,sodium citrate hydrochloric acid, sodium hydroxide,tris(hydroxymethyl)aminomethane,bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane, and sodium hydrogencarbonate and others known to those of ordinary skill in the art.Buffers are commonly used to adjust the pH to a desirable range forintraperitoneal use. Usually a pH of around 5 to 9, 5 to 8, 6 to 7.4,6.5 to 7.5, or 6.9 to 7.4 is desired.

The suspension can comprise one or more demulcents. A demulcent is anagent that forms a soothing film over a mucous membrane, such as themembranes lining the peritoneum and organs therein. A demulcent mayrelieve minor pain and inflammation and is sometimes referred to as amucoprotective agent. Suitable demulcents include cellulose derivativesranging from about 0.2 to about 2.5% such as carboxymethylcellulosesodium, hydroxyethyl cellulose, hydroxypropyl methylcellulose, andmethylcellulose; gelatin at about 0.01%; polyols in about 0.05 to about1%, also including about 0.05 to about 1%, such as glycerin,polyethylene glycol 300, polyethylene glycol 400, and propylene glycol;polyvinyl alcohol from about 0.1 to about 4%; povidone from about 0.1 toabout 2%; and dextran 70 from about 0.1% when used with anotherpolymeric demulcent described herein.

The suspension can comprise one or more alkalinizing agents to adjustthe pH. As used herein, the term “alkalizing agent” is intended to meana compound used to provide an alkaline medium. Such compounds include,by way of example and without limitation, ammonia solution, ammoniumcarbonate, potassium hydroxide, sodium carbonate, sodium bicarbonate,and sodium hydroxide and others known to those of ordinary skill in theart

The suspension can comprise one or more acidifying agents to adjust thepH. As used herein, the term “acidifying agent” is intended to mean acompound used to provide an acidic medium. Such compounds include, byway of example and without limitation, acetic acid, amino acid, citricacid, nitric acid, fumaric acid and other alpha hydroxy acids,hydrochloric acid, ascorbic acid, and nitric acid and others known tothose of ordinary skill in the art.

The suspension can comprise one or more antifoaming agents. As usedherein, the term “antifoaming agent” is intended to mean a compound orcompounds that prevents or reduces the amount of foaming that forms onthe surface of the fill composition. Suitable antifoaming agents includeby way of example and without limitation, dimethicone, SIMETHICONE,octoxynol and others known to those of ordinary skill in the art.

The suspension can comprise one or more viscosity modifiers thatincrease or decrease the viscosity of the suspension. Suitable viscositymodifiers include methylcellulose, hydroxypropyl methycellulose,mannitol and polyvinylpyrrolidone.

In some embodiments, the chemotherapeutic particle is present in asuspension further comprising a polysorbate. In one specific embodiment,the chemotherapeutic particle is present in a suspension furthercomprising a polysorbate, wherein the polysorbate is polysorbate 80. Inother embodiments, the polysorbate or polysorbate 80 is present in thesuspension at a concentration of between about 0.01% v/v and about 1.5%v/v. The inventors have surprisingly discovered that the recited verysmall amounts of polysorbate 80 reduce the surface tension at theinterface of the chemotherapeutic particles and the aqueous carrier inthe suspension (such as saline). These embodiments are typicallyformulated near the time of use of the composition. In theseembodiments, the particles are not coated with the polysorbate orpolysorbate 80. In various other embodiments, the polysorbate orpolysorbate 80 is present in the suspension at a concentration ofbetween about 0.01% v/v and about 1% v/v, about 0.01% v/v and about 0.5%v/v, about 0.01% v/v and about 0.4% v/v, about 0.01% v/v and about 0.25%v/v, about 0.05% v/v and about 0.5% v/v, about 0.05% v/v and about 0.25%v/v, about 0.1% v/v and about 0.5% v/v, about 0.1% v/v and about 0.25%v/v, about 0.1% v/v, about 0.16 v/v, or about 0.25% v/v. In furtherembodiments, the chemotherapeutic, such as paclitaxel, is present in thesuspension at a concentration between about 1 mg/ml and about 40 mg/ml,or about 6 mg/ml and about 20 mg/ml. In various further embodiments, thechemotherapeutic is present in the suspension at a concentration betweenabout 6 mg/ml and about 15 mg/ml, between about 6 mg/ml and about 10mg/ml, about 10 mg/ml and about 20 mg/ml, about 10 mg/ml and about 15mg/ml, about 6 mg/ml, about 10 mg/ml, or about 15 mg/ml. In variousfurther embodiments, the aqueous carrier in the composition may besaline, such as about 0.9% sodium chloride.

The present invention thus also provides a suspension comprising:

(a) chemotherapeutic particles;

(b) a pharmaceutically acceptable carrier; and

(c) a polysorbate, wherein the polysorbate is present in the suspensionat a concentration of between about 0.01% v/v and about 1.5% v/v, orbetween about 0.01% v/v and about 1% v/v, about 0.01% v/v and about 0.5%v/v, about 0.01% v/v and about 0.4% v/v, about 0.01% v/v and about 0.25%v/v, about 0.05% v/v and about 0.5% v/v, about 0.05% v/v and about 0.25%v/v, about 0.1% v/v and about 0.5% v/v, about 0.1% v/v and about 0.25%v/v, about 0.1% v/v, about 0.16 v/v, or about 0.25% v/v.

In one embodiment, the polysorbate is polysorbate 80. In one embodiment,the pharmaceutically acceptable carrier is 0.9% saline solution. Inanother embodiment, the chemotherapeutic particles are taxane particles.

The present invention thus also provides kits, comprising:

(a) a first vial containing chemotherapeutic particles;

(b) a second vial containing a polysorbate and a pharmaceuticallyacceptable carrier; and

(c) instructions for reconstituting the chemotherapeutic particles intoa suspension by combining the contents of the first vial and the secondvial, and for diluting the suspension with a diluent solution, such as0.9% saline solution, prior to administration to a patient.

In one embodiment, the polysorbate is polysorbate 80. In one embodiment,the pharmaceutically acceptable carrier is 0.9% saline solution. In oneembodiment, the contents of the first and second vial are sterile. Whenthe suspension of chemotherapeutic particles and polysorbate is dilutedwith the diluent solution, excessive dissolution of the chemotherapeuticparticles is prevented.

The compositions, suspensions, and kits of this aspect of the inventioncan include any embodiment or combination of embodiments of thechemotherapeutic particles described herein, and any embodiment of thepolysorbate or polysorbate 80 concentration described herein. In variousfurther embodiments, the aqueous carrier in the composition may besaline, such as about 0.9% sodium chloride. The compositions,suspensions, and kits can exclude polymers, proteins (such as albumin),polyethoxylated castor oil, and/or polyethylene glycol glyceridescomposed of mono-, di- and triglycerides and mono- and diesters ofpolyethylene glycol.

The compositions and kits may further comprise other components asappropriate for a given chemotherapeutic particle. In one embodiment, adocetaxel particle may further comprise ethanol as a solvent; anysuitable amount of ethanol may be used, such as between about 0.13% and3.2% Dehydrated Alcohol (Ethanol), 200 proof, Undenatured, USP.

The subject may be any suitable subject that can benefit from thetreatment, including but not limited to mammals (such as humans andother primates, dogs, cats, horses, cattle, pigs, sheep, goats, etc.)

The “amount effective” of the chemotherapeutic particle can bedetermined by an attending physician based on all relevant factors. Thechemotherapeutic particles may be the sole chemotherapeuticadministered, or may be administered with other therapeutics as deemedappropriate by an attending physician in light of all circumstances. Inone embodiment, the methods further comprise treating the subject withthe standard of care for the tumor being treated, such as intravenouschemotherapy, radiation therapy radiotherapy, surgical resection, etc.For example, the methods for treating prostate cancer may be combinedwith one or more of salvage prostatectomy, focal or whole-glandbrachytherapy, cryotherapy, high-intensity focused ultrasound (HIFU),and stereo-tactic body radiotherapy (SBRT).

Direct injection of the chemotherapeutic particles into the tumor may beaccomplished by any suitable means. In non-limiting embodiments, theinjection may be carried out via magnetic resonance imaging-transrectalultrasound fusion (MR-TRUS) guidance (such as for injecting prostatetumors), or via endoscopic ultrasound-guided fine needle injection(EUS-FNI).

Example 1. Production of Paclitaxel and Docetaxel Particles

Materials and Methods

Raw paclitaxel and docetaxel were purchased from Phyton Biotech (BritishColumbia, Canada), lot number FP2-15004 and DT7-14025, respectively.Both were characterized in their raw form. The milling of both drugs wasaccomplished using a Deco-PBM-V-0.41 mill (Deco). The milling conditionsfor both compounds were as follows:

Ball size=5 mm

RPM=600

Processing time=60 min

Room temperature.

Preparation of Paclitaxel Particles

A solution of 65 mg/ml of paclitaxel was prepared in acetone. A BETEMicroWhirl® fog nozzle (BETE Fog Nozzle, Inc) and a sonic probe(Qsonica, model number Q700) were positioned in the crystallizationchamber approximately 8 mm apart. A stainless steel mesh filter withapproximately 100 nm holes was attached to the crystallization chamberto collect the precipitated paclitaxel nanoparticles. The supercriticalcarbon dioxide was placed in the crystallization chamber of themanufacturing equipment and brought to approximately 1200 psi at about38° C. and a flow rate of 24 kg/hour. The sonic probe was adjusted to60% of total output power at a frequency of 20 kHz. The acetone solutioncontaining the paclitaxel was pumped through the nozzle at a flow rateof 4.5 mL/minute for approximately 36 hours. Paclitaxel nanoparticlesproduced had an average number-weighted mean size of 0.81 μm with anaverage standard deviation of 0.74 μm over three separate runs.

Preparation of Docetaxel Particles

A solution of 79.32 mg/ml of docetaxel was prepared in ethanol. Thenozzle and a sonic probe were positioned in the pressurizable chamberapproximately 9 mm (apart. A stainless steel mesh filter withapproximately 100 nm holes was attached to the pressurizable chamber tocollect the precipitated docetaxel nanoparticles. The supercriticalcarbon dioxide was placed in the pressurizable chamber of themanufacturing equipment and brought to approximately 1200 psi at about38° C. and a flow rate of 68 slpm. The sonic probe was adjusted to 60%of total output power at a frequency of 20 kHz. The ethanol solutioncontaining the docetaxel was pumped through the nozzle at a flow rate of2 mL/minute for approximately 95 minutes). The precipitated docetaxelagglomerates and particles were then collected from the supercriticalcarbon dioxide as the mixture is pumped through the stainless steel meshfilter. The filter containing the nanoparticles of docetaxel was openedand the resulting product was collected from the filter.

Docetaxel nanoparticles produced had an average number-weighted meansize of 0.82 μm with an average standard deviation of 0.66 μm over threeseparate ethanol runs.

Particle Size Analysis

Particle size was analyzed by both light obscuration and laserdiffraction methods. An Particle Sizing Systems AccuSizer 780 SIS systemwas used for the light obscuration method and Shimadzu SALD-7101 wasused for the laser diffraction method. Paclitaxel nanoparticles wereanalyzed using 0.10% (w/v) sodium dodecyl sulfate (SDS) in water as thedispersant. Docetaxel nanoparticles were analyzed using isopar G as thedispersant.

Paclitaxel suspensions were prepared by adding approximately 7 mL offiltered dispersant to a glass vial containing approximately 4 mg ofpaclitaxel particles. The vials were vortexed for approximately 10seconds and then sonicated in a sonic bath approximately 1 minute. Ifthe sample was already suspended, 1:1 solution of paclitaxel suspensionto 0.1% SDS solution was made, vortexed for 10 seconds, and sonicated inthe sonic bath for 1 minute.

Docetaxel suspensions were prepared by adding approximately 7 mL offiltered dispersant to a plastic vial containing approximately 4 mg ofdocetaxel particles. The vial was vortexed for approximately 10 secondsand then sonicated in a sonic bath for approximately 2 minutes. Thissuspension was used for laser diffraction analysis. Unused suspensionwas poured into a 125 mL particle-free plastic bottle, which was thenfilled to approximately 100 mL with filtered dispersant. The suspensionwas vortex for approximately 10 seconds and then sonicated in the sonicbath for approximately 2 minutes. This diluted suspension was used forlight obscuration analysis.

A background test was first performed prior to analyzing particles onthe AccuSizer 780 SIS. A new particle-free plastic bottle was filledwith blank suspension solution by pumping from a reservoir, using aperistaltic pump, through a 0.22 μm Millipore filter and into thebottle. A background analysis was run to ensure the particle/mL countwas below 100 particles/mL. A small amount of paclitaxel suspension,5-100 μL, depending upon concentration of solution, was pipetted intothe plastic bottle in place from the background test and was filled with˜100 mL dispersant and the analysis was started. Counts were monitoredand paclitaxel solution added to reach and/or maintain 6000-8000particle counts/mL during the entire analysis. Once the analysis wascompleted, the background data was removed and any measurement with lessthan four counts was removed.

To analyze particles on SALD-7101 using a batch cell, the analysis wasstarted by choosing Manual Measurement. The refractive index was set as1.5 to 1.7. The batch cell was filled with filtered dispersant just pastthe etched line. The blank measurement was ran. A small amount of API(paclitaxel or docetaxel) suspension was pipetted, generally <1 mL,depending upon concentration of solution as low as 100 μL, into thebatch cell as needed to achieve an acceptable absorbance between 0.15and 0.2 absorbance units. The measurements were executed, and theresulting graph with the highest level of confidence was selected;background was automatically accounted for.

BET Analysis

A known mass between 200 and 300 mg of the analyte was added to a 30 mLsample tube. The loaded tube was then mounted to a Porous Materials Inc.SORPTOMETER®, model BET-202A. The automated test was then carried outusing the BETWIN® software package and the surface area of each samplewas subsequently calculated.

Bulk Density Analyte

Paclitaxel or docetaxel particle preparations were added to a 10 mLtared graduated cylinder through a plastic weigh funnel at roomtemperature. The mass of the drug was measured to a nearest 0.1 mg, thevolume was determined to the nearest 0.1 mL and the density calculated.

Dissolution Studies

Paclitaxel

Approximately 50 mg of material (i.e.: raw paclitaxel, milledpaclitaxel, or paclitaxel particles) were coated on approximately 1.5grams of 1 mm glass beads by tumbling the material and beads in a vialfor approximately 1 hour. Beads were transferred to a stainless steelmesh container and placed in the dissolution bath containingmethanol/water 50/50 (v/v) media at 37° C., pH 7, and a USP Apparatus II(Paddle), operating at 75 rpm. At 10, 20, 30, 60, and 90 minutes, a 5 mLaliquot was removed, filtered through a 0.22 μm filter and analyzed on aU(V/V) is spectrophotometer at 227 nm. Absorbance values of the sampleswere compared to those of standard solutions prepared in dissolutionmedia to determine the amount of material dissolved.

Docetaxel

Approximately 50 mg of material (i.e.: raw docetaxel, milled docetaxel,or docetaxel particles) was placed directly in the dissolution bathcontaining methanol/water 15/85 (v/v) media at 37° C., pH 7, and a USPApparatus II (Paddle), operating at 75 rpm. At 5, 15, 30, 60, 120 and225 minutes, a 5 mL aliquot was removed, filtered through a 0.22 μmfilter, and analyzed on a UV/VIS spectrophotometer at 232 nm. Absorbancevalues of the samples were compared to those of standard solutionsprepared in dissolution media to determine the amount of materialdissolved.

Results

The BET surface area of particles produced using the above protocol andvariations thereof (i.e.: modifying nozzles, filters, sonic energysources, flow rates, etc.) ranged between 22 and 39 m²/g. By comparison,the BET surface area of raw paclitaxel was measured at 7.25 m²/g (FIG.2), while paclitaxel particles made according to the methods of U.S.Pat. Nos. 5,833,891 and 5,874,029 ranged from 11.3 to 15.58 m²/g.Exemplary particle sizes produced using the methods of the invention areshown in Table 1.

TABLE 1 Surface Mean Size St Dev area μm μm m²/g Number Volume NumberVolume 1 38.52 0.848 1.600 0.667 0.587 2 33.82 0.754 0.988 0.536 0.486 335.90 0.777 1.259 0.483 0.554 4 31.70 0.736 0.953 0.470 0.466 5 32.590.675 0.843 0.290 0.381 6 38.22 0.666 0.649 0.344 0.325 7 30.02 0.6700.588 0.339 0.315 8 31.16 0.672 0.862 0.217 0.459 9 23.90 0.857 1.5600.494 0.541 10 22.27 0.857 1.560 0.494 0.541 11 26.19 0.861 1.561 0.4650.546

Comparative studies on bulk density, SSA, and dissolution rates (carriedout as noted above) for raw drug, milled drug particles, and drugparticles produced by the methods of the present invention are providedin Tables 2 and 3 below. The full dissolution time course for thepaclitaxel and docetaxel materials are provided in Tables 4 and 5,respectively.

TABLE 2 Compound: Paclitaxel Raw Particles Characteristic Material Batch1 Batch 2 Mean Milled Number Mean (um) 1.16 0.83 0.67 0.75 0.89 VolumeMean (um) 1.29 1.42 0.57 1.00 1.35 Bulk Density (g/cm³) 0.26 0.060 0.110.085 0.31 Surface Area (m²/g) 10.4 35.6 39.8 37.7 15.0 Dissolution (30min) 18% 42% 52% 47% 32%

TABLE 3 Compound: Docetaxel Raw Particles Characteristic Material Batch1 Batch II Mean Milled Number Mean (um) 1.58 0.92 0.80 0.86 1.11 VolumeMean (um) 5.05 4.88 4.03 4.46 3.73 Bulk Density (g/cm³) 0.24 0.062 0.0960.079 0.44 Surface Area (m²/g) 15.9 43.0 45.4 44.2 15.2 Dissolution (30min) 11% 27% 27% 27% 9%

TABLE 4 Paclitaxel Dissolution time course Timepoint Milled (minutes)Paclitaxel Raw Material Paclitaxel Particles Paclitaxel 0 0.0% 0.0% 0.0%10 14.0% 40.2% 23.0% 20 17.8% 47.6% 30.0% 30 18.4% 51.9% 32.3% 60 23.9%58.3% 38.6% 90 28.6% 62.9% 43.5%

TABLE 5 Docetaxel Dissolution time course Timepoint Milled (minutes)Docetaxel Raw Material Docetaxel Particles Docetaxel 0 0.0% 0.0% 0.0% 53.2% 12.1% 3.2% 15 6.9% 21.7% 5.9% 30 11.2% 27.2% 9.3% 60 16.4% 32.9%12.2% 120 22.4% 38.9% 13.6% 225 26.8% 43.1% 16.0%

Example 2. Pharmacokinetics and Tissue Distribution of PaclitaxelParticles Following Intraperitoneal Injection in Mice

Purpose: This study was conducted to determine the level of absorptionof paclitaxel from the peritoneal cavity into the systemic circulationfollowing intraperitoneal delivery of a paclitaxel particle suspension.Tissue distribution of paclitaxel from the paclitaxel particlesuspension following intraperitoneal administration was also evaluated.

Experimental Details: Female C57BL6 mice were inoculated with ID8ovarian cancer cells and tumors were allowed to grow for 45 days. Thesemice were treated with paclitaxel particle suspension (36 mg/kg) in 0.9%saline via intraperitoneal administration in a total volume of 4 mL.Plasma and peritoneal fluid samples were collected at Time zero(pre-dose), 1, 6, 24 and 48 hours (at least four mice per time point)and paclitaxel in the plasma and peritoneal fluid was measured byLCMSMS. In addition, tissue samples were collected at Time zero(pre-dose), 1, 6, 24 and 48 hours post intraperitoneal administration ofpaclitaxel particles. Inguinal lymph nodes, peritoneal wall, ovary,liver, heart, kidney, lung, brain and tumor tissue samples from miceadministered paclitaxel particles were analyzed by LC/MS/MS.

Results and Significance: The results of the paclitaxel levels in theplasma, peritoneal fluid and organ tissue samples are shown in thefollowing table. Plasma paclitaxel remained at a very low level over the48 hour period. The paclitaxel levels in the peritoneal fluid were muchhigher and demonstrated a substantial amount of variation. The limit ofquantitation of the analytical method for paclitaxel was 0.01 μg/gm. Thelevels of paclitaxel in tissues inside the peritoneal cavity wereconsistently high as demonstrated by the results for the ovarian tumors,ovary, inguinal lymph nodes and peritoneal membrane. In contrast, thepaclitaxel levels in tissues outside the peritoneal cavity wereconsistently lower as shown in the liver, heart, lung and brain tissues.These same results are shown in Table 6.

This data is significant because of the unexpectedly high levels ofpaclitaxel in the tissues that are in contact with the peritoneal fluid(ovarian tumors, ovary, inguinal lymph nodes and peritoneal membrane),and little paclitaxel to tissues not in contact with the peritonealfluid. Based on these and other studies, the release of paclitaxel fromthe paclitaxel particles continues for several weeks and would beexpected to provide a continuously high amount of paclitaxel, whichwould mean that the paclitaxel would accumulate in very high levels ifinjected directly into the tumor.

TABLE 6 Summary Levels of Paclitaxel in chemotherapeuticparticles-treated Mouse Tissue, Plasma and Peritoneal Fluid (values inμg/g) (4 animals) Time Tumor Ovary Lymph Membrane Liver Heart Plasma IPFluid Lung Brain (hours) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g)(μg/ml) (μg/ml) (μg/g) (μg/g) 0 0.0 0.00 0.00 0 0 0 0.0 0.0 0.00 0 0.00.00 0.00 0 0 0 0.0 0.0 0.00 0 0.0 0.00 0.00 0 0 0 0.0 0.0 0.00 0 0.00.00 0.00 0 0 0 0.0 0.0 0.00 0 1 458.6 500.00 190.80 107.23 7.0 0 0.549.0 1.97 0.04 180.1 263.64 199.28 31.42 6.7 0 0.0 1.0 BQL 0.13 135.3498.00 122.90 9.38 3.1 0 0.0 0.7 2.78 BQL 158.1 1193.75 296.36 133.47 90 0.2 24.6 0.78 0.36 6 126.2 1068.97 486.87 64.8 48.8 0 0.0 1.2 1.180.11 240.0 816.33 648.15 91.38 46.6 0 0.4 40.4 1.65 BQL 701.2 751.0548.86 132.35 27.0 0 0.0 1.8 0.75 BQL 89.7 211.20 143.33 97.41 14.3 0 0.04.2 1.56 0.08 24 81.3 502.70 90.14 27.09 41.3 0 0.0 1.5 5.22 0.29 204.51706.42 86.83 65.31 41.9 2.77 0.2 24.0 0.92 0.08 241.2 335.58 238.64109.96 50.3 0 0.0 5.1 0.83 1.36 208.8 603.64 254.00 124.41 29.7 3 BQ BQ1.10 0.82 48 294.0 529.94 124.17 270.95 116.4 0 0.1 12.0 BQL 0.05 4001389.83 1795.45 79.5 44.2 0 0.0 1.5 BQL 0.06 505.4 711.48 81.94 80.3376.6 0 0.0 5.2 BQL 0.18 174.9 1272.11 224.88 218.24 34.0 0 0.2 28.6 BQLBQL

Example 3. Glioblastoma Study

In this study, we assessed the efficacy of paclitaxel particles againstglioblastomas (GB). Nude mouse brains were injected with GB cells toestablish primary tumors, which were injected after two weeks withpaclitaxel particles. We monitored survival benefit against a controlgroup that received only saline injections and a control group that hasreceived Taxol™ (formulated in cremophor) injections. We delivered adose of 100 mg/m² to the growing tumor by direct injection. The tablebelow shows four different tumor sizes and the corresponding dose ofpaclitaxel particles of the invention, assuming a spherical shape forthe tumor. As a control experiment Taxol™ formulated in cremophor anddiluted in saline to the correct concentration was used.

TABLE 7 Tumor Tumor ng of Nanotax Dilution Radius (mm) Surface (mm²) perinjection* Factor** 0.5 3.1 314.2 50 1 12.6 1256.6 13 1.5 28.3 2827.4 62 50.3 5026.5 3 *At a dose of 100 mg/m² **5 μl injection; Nanotax stocksolution: 3,150 ng/μl

At the highest dose of 5 μg of Paclitaxel per injection no toxicity wasobserved. Injected mice were kept alive for 8 to 9 days, after which noneurological symptoms were observed. After 9 days the mice weresacrificed and the brains harvested; the brains were dissected along theinjection path and analyzed. Neither the paclitaxel particle group northe Taxol™ group showed necrosis or lesions. Brain slices were usedwithout further preparation to be analyzed with three different imagingtechnologies to create a composite image (data not shown):

(a) 2nd harmonic generation (SHG): Images appear in blue, mostlycollagen and vessels and paclitaxel particles.

(b) Two-photon excitation fluorescence (TPEF): Images appear in green,mostly reactive cells, microglia, macrophages, some neuronal bodies.

(c) Coherent Anti-Stokes Raman Scattering (CARS): Images appear in red,tuned to —CH2 vibrations to look at lipids—mostly to myelin in the CNS,but also lipid droplet containing cells called foam cells will give apositive signal. Degenerating cells and macrophages with lipid vacuoleswill also show up.

Due to the non-linear optical properties of the paclitaxel particlecrystals the crystals can be seen directly with the second harmonicgeneration imaging technology. Clusters of paclitaxel particles wereclearly visible at the injection site (i.e.: accumulated within thetumor) 9 days after injection in a mouse that was injected with 5 μg ofpaclitaxel particles and showed no neurological symptoms (data notshown).

Example 4. Prostate Cancer Studies

Two in vivo nonclinical pharmacology studies were conducted to determinethe effects of intratumoral (ITU) delivery of paclitaxel particles in anude mouse solid tumor PC-3 human prostate carcinoma nude mousexenograft. In Study Number P-PPr-01-2015 mice were administeredpaclitaxel particles (also referred to as NanoPac™) at 37.5 mg/kg, qwk×1(i.e.: a single dose); 12.5 mg/kg, qwk×3 (i.e.: a single dose threetimes per week); or 37.5 mg/kg, qwk×3; paclitaxel (30 mg/kg, qwk×3), orvehicle (0.1% w/v Polysorbate 80 in saline, qwk×3). Treatments withpaclitaxel particles and vehicle were by ITU injection. Thirty-five daysafter cell implant, on D1 of the study, animals with individual tumorvolumes of 108 to 196 mm3 were sorted into five groups (n=10) with groupmean tumor volumes ranging from 150-153 mm3. All treatments wereinitiated on D1 of the study. Paclitaxel was administered as an IVinfusion. All experimental treatments were tested in groups of 10 mice.Tumors were measured twice per week. Partial treatment outcome was basedon percent tumor growth inhibition (% TGI), defined as the percentdifference between the Day 32 median tumor volumes of treated andvehicle-control mice. The end of the experiment was a tumor volume of2000 mm³ or 60 days, whichever came first.

Paclitaxel particles were significantly active in the PC-3 prostatecarcinoma model (FIG. 1). Treatment of animals with either a single doseor three weekly doses of paclitaxel particles resulted in identicalsurvival extension and an increased number of studysurvivors/regressions when compared to vehicle-treated control animals.There was not a dose dependent response as dosing with paclitaxelparticles at 12.5 or 37.5 mg/kg qwk×3 produced the maximum survivalextension attainable in the study. Results for all paclitaxelparticle-treated animals were well above the 60% TGI thresholdindicative of potential therapeutic activity. IV-administered paclitaxelproduced significant TGI and significantly improved survival extensionin this study. All treatments were well-tolerated.

Study Number PD-PPr-02-2016 included docetaxel particles (also referredto as NanoDoce™) made as described above, as well as paclitaxelparticles. Mice with PC-3 prostate carcinoma tumor xenografts wereadministered by ITU injection docetaxel particles at 100 mg/kg, qwk×1;37.5 mg/kg, qwk×3; or 100 mg/kg, qwk×3; paclitaxel particles at 37.5mg/kg, qwk×3; or vehicle (0.1% w/v Polysorbate 80 in saline, qwk×3) viaIT injection; or were intravenously administered docetaxel (30 mg/kg,qwk×3). Twenty-six days after cell implant, on D1 of the study, animalswith individual tumor volumes of 108 to 172 mm3 were sorted into fivegroups (n=10) with group mean tumor volumes ranging from 136-141 mm3.All treatments were initiated on D1 of the study. All experimentaltreatments were tested in groups of 10 mice. Tumors were measured twiceper week. The end of the experiment was a tumor volume of 2000 mm³ or 60days, whichever came first. The maximum possible tumor growth delay(TGD) in this study was determined to be 41%.

Paclitaxel particles and docetaxel particles were highly active in thePC-3 prostate carcinoma model. All regimens, regardless of thedose/schedule, produced significant survival extension achieving in allcases the maximum possible TGD allowable in the study. Results foranimals treated with paclitaxel particles, docetaxel particles, anddocetaxel demonstrated similar levels of efficacy. All treatmentregimens were well-tolerated. FIG. 2 shows mean tumor volume for mousePC-3 prostate tumor xenografts treated IT with vehicle, NanoDoce,NanoPac and treated IV with docetaxel.

Example 5. MDA-MB-231 Breast Cancer Studies

The objective of this study was to determine the response of thesubcutaneously (SC)-implanted MDA-MB-231 human breast cancer xenograftsto treatment with paclitaxel particles (“NanoPac™”) and docetaxelparticles (“NanoDoce™”). Test articles were as shown in Table 8

TABLE 8 Dose Test Dose Conc. Dose Group Article Vehicle (mg/kg) (mg/mL)Volume 1 Vehicle 0.1% T80/99.9% Sodium Chloride for 0 0 0.063* mLInjection USP (0.9%) 2 NanoPac ™ 0.1% T80/99.9% Sodium Chloride for 10040 0.063* mL Injection USP (0.9%) 3 NanoDoce ™ 0.266% T80/2.66%ethanol/97.07% 100 40 0.063* mL Sodium Chloride for Injection USP (0.9%)4 Docetaxel 7.5% ethanol/7.5% T80/85% Sodium 30 3    10 mL/kg Chloridefor Injection USP (0.9%) 5 Paclitaxel 12.5% ethanol/12.5% Cremophor 30 3   10 mL/kg EL/75% Sodium Chloride for Injection USP (0.9%) *Based on a25 g mouseDose Formulation

NanoPac™: 0.75 mL of the 1% Polysorbate 80 (“T80”) reconstitutionsolution was added into the vial of NanoPac™ (306 mg/vial). Thereconstitution solution was prepared, sterile filtered through a 0.22micron filter on the day of preparation, and stored refrigerated (2 to8° C.) for no more than 7 days. The vial was vigorously hand shaken withinversion for 1 minute. Immediately after shaking, 6.9 mL of 0.9% SodiumChloride for Injection USP was added to the vial to make a 40 mg/mLsuspension (7.65 mL) and hand shaken the vial for another 1 minute.After mixing, the suspension was allowed to sit undisturbed for 5minutes to reduce entrapped air and foam.

Handling: Room temperature.

Stability: Dose formulations were maintained at room temperature andconsidered stable for 8 hours.

NanoDoce™: 1.33 ml of the 1% T80/10% ethanol/in 0.9% salinereconstitution solution was added into the vial of NanoDoce particles(200 mg/vial). The reconstitution solution was prepared, sterilefiltered through a 0.22 micron filter on the day of preparation, andstored refrigerated (2 to 8° C.) for no more than 7 days. The vial wasvigorously hand shaken with inversions for 1 minute. Immediately aftershaking, 2 ml of 0.9% Sodium Chloride for Injection USP was added andthe vial was hand shaken for another 1 minute. An additional 1.67 ml of0.9% Sodium Chloride for Injection USP was added to the vial and handmixing with inversion continued for 1 minute. Final volume was 5 mL of a40 mg/mL suspension. After mixing, the suspension was allowed to situndisturbed for at least 5 minutes to reduce entrapped air and foam.

Handling: Room temperature.

Stability: Dose formulations were maintained at room temperature andconsidered stable for 8 hours.

Paclitaxel: Bulk Paclitaxel was added to the clinical formulation ofPaclitaxel (6 mg/mL in 50% ethanol: 50% Cremophor EL) to make a 12 mg/mLsolution and vortexed as needed. Sodium Chloride for Injection USP(0.9%) was added to make a 3 mg/mL solution of Paclitaxel and vortexedto mix.

Handling: Warm water.

Stability: Injected within 20 minutes of formulation.

Docetaxel: Sodium Chloride for Injection USP (0.9%) was added to make a3 mg/mL solution of Docetaxel (20 mg/mL in 50% ethanol:50% T80) andvortexed to mix.

Handling: Room temperature.

Stability: Dose formulations were maintained at room temperature andconsidered stable for 8 hours.

Test System:

Species & Strain: Mouse; NCr-nu/nu

Supplier: Charles River Laboratories

Number on Study: Females—50

Study Design:

A total of 88 female NCr-nu/nu mice were implanted with MDA-MB-231breast tumor fragments from an in vivo passage subcutaneously (SC) onthe right flank. The day of tumor cell implantation (Aug. 4, 2016) wasdesignated as Day 0. Individual tumors of 50 animals grew to 100-221 mgin weight (100-221 mm³ in size) on Day 13 after tumor cell implantation(Aug. 17, 2016), the day of treatment initiation/stage day (SD). Fiftyanimals were assigned to Groups 1-5 such that the mean tumor weights inall five groups on Day 13 were 147.4-149.6 mg (median tumor weights were135-149 mg).

TABLE 9 No. Dose Total Dose of Dose Route/ Dose Conc. Dose DeliveredGroup mice Test Article Schedule (mg/kg) (mg/mL) Volume (mg) 1 10Vehicle IT*/qwk × 3 — — 0.063 mL 0 2 10 NanoPac ™ IT*/qwk × 3 100 400.063 mL 7.56 3 10 NanoDoce ™ IT*/qwk × 3 100 40 0.063 mL 7.56 4 10Docetaxel IV/qwk × 3 30 3   10 mL/kg 2.25** 5 10 Paclitaxel IV/qwk × 330 3   10 mL/kg 2.25** *IT = Intratumoral **The total dose was based ona 25 g mouse.

The NanoPac™ and NanoDoce™ are suspensions. Prior to filling eachsyringe and between the injections of each animal, the test article wasgently swirled/inverted. Intratumoral administration of the testmaterial entailed the use of six needle tracks per tumor (27G ½ inchneedle). One sixth of the dose volume was injected as a slow bolus ineach track, with ˜15 seconds pause before removing the needle slowly,and moving to the next injection site spaced over the surface of thetumor. The animals were observed once daily for mortality andmoribundity. The mice were weighed and the tumor measurements were takentwice weekly starting on the first day of treatment. Length and widthwere measured for each tumor. Tumor volume was determined using theformula for an ellipsoid sphere:Length×Width^(2/)2=Volume (mm³)

This formula was also used to calculate tumor weight, assuming unitydensity (1 mm³=1 mg). Limit of tumor detection was 32 mg (4×4 mm). Theexperiment lasted for 61 days from the day of tumor implant. Animalswhose weight decreased more than 30% from the weight on the first day oftreatment or whose tumor reached 4,000 mg in weight, ulcerated, orsloughed off was euthanized prior to study termination. Comparisonsbetween mean tumor volume after administration of test articlesDocetaxel and NanoDoce™ and between Paclitaxel and NanoPac™ arepresented in FIG. 3. More than 50% of animals in groups 1-2 were lostdue to tumor ulceration, death, or large tumor size by day 21, and thusthe mean data for these groups is not presented.

Results

The human MDA-MB-231 breast cancer xenografts in the vehicle-treatedcontrol group (Group 1) grew in all 10 mice to a mean tumor weight of1,929 mg on Day 29 (n=9; the first measurement after the last day oftreatment, 1 animal was removed on Day 22 due to ulceration). There wereno animal deaths or mean body weight loss. Tumor inhibition was onlycompared to the control until Day 33 when there were 6 animals in thecontrol group.

There were three animal deaths (Day 20 and 27) with three IT treatments(weekly) of NanoPac™ at a dose of 100 mg/kg (Group 2) and no mean bodyweight loss. The animal deaths occurred on days of IT injections. Therepeated NanoPac™ treatment did not significantly inhibit the growth ofthe MDA-MB-231 breast cancer xenografts [mean tumor weight of 1,031 mgon Day 29 (n=7) corresponding to a % difference of −46.6% relative tothe vehicle-treated control (p>0.05)]. The nadir of response occurred onDay 26 with a % difference of −51.8% which was significantly differentthan the vehicle-treated control group (p<0.05); Day 19 was alsosignificantly different than the vehicle control group (p<0.05). Therewere two complete tumor regressions which remained tumor-free at the endof the study.

There were two animal deaths (Day 20 and 27) with the three ITtreatments (weekly) of NanoDoce™ at a dose of 100 mg/kg (Group 3) and nomean body weight loss. The animal deaths occurred on days of ITinjections. The repeated NanoDoce™ treatment significantly inhibited thegrowth of the MDA-MB-231 breast cancer xenografts [mean tumor weight of143 mg on Day 29 (n=8) corresponding to a % difference of −92.6%relative to the vehicle-treated control (p<0.05)]. The tumor growthinhibition progressed to the nadir on Day 33 with a % difference of−94.2% (p<0.05); the mean tumor volume decreased to 0 on Day 40. The %difference was significant starting on Day 19 until no comparison waspossible. The eight surviving animals remained tumor-free at the end ofthe study on Day 61.

There were no animal deaths following the three IV treatments (weekly)of docetaxel at a dose of 30 mg/kg (Group 4) with a mean body weightloss of 19.6% (4.78 g, Day 33). The repeated docetaxel treatmentsignificantly inhibited the growth of the MDA-MB-231 breast cancerxenografts [mean tumor weight of 16 mg on Day 29 (n=10) corresponding toa % difference of −99.2% relative to the vehicle-treated control(p<0.001)]. The tumor growth inhibition progressed to the nadir on Day33 with a % difference of −99.8% (p<0.05); the mean tumor volumedecreased to 0 on Day 40. The % difference was significant starting onDay 19 until no comparison was possible. Complete tumor inhibition wasobserved on Day 40 when all 10 animals had completely regressed tumorswhich remained tumor-free at the end of the study. The docetaxel treatedanimals' tumor growth inhibition was significantly different than theNanoDoce™ treated animals' from Days 22-33 only.

Three IV treatments (weekly) with paclitaxel at a dose of 30 mg/kg(Group 5) were tolerated without animal deaths and no mean body weightloss. The paclitaxel treatment significantly inhibited the growth of theMDA-MB-231 breast cancer xenografts [mean tumor weight of 118 mg on Day29 (n=10) corresponding to a % difference of −93.9% relative to thevehicle-treated control (p<0.05)]. The nadir of response occurred on Day33 with a % difference of −97.4%. The % difference was significantstarting on Day 19 until no comparison was possible. There were sixcomplete tumor regressions with three tumor-free animals at the end ofthe study. The paclitaxel treated animals' tumor growth inhibition wassignificantly different than the NanoPac™ treated animals' Days 15 and22-36.

Discussion

There were three or two animals dead in groups treated IT with NanoPac™or NanoDoce™, respectively. The animal deaths occurred early and werenot considered to be treatment related. The control group and treatmentwith paclitaxel or docetaxel was tolerated without animal deaths. Growthof the human MDA-MB-231 breast cancer xenografts was significantlyinhibited by treatment with NanoDoce™ which had eight tumor-freesurviving animals. Growth of the human MDA-MB-231 breast cancerxenografts was inhibited by treatment with docetaxel which had 10tumor-free animals and was significantly different than NanoDoce™treatment on Days 22-33. Growth of the human MDA-MB-231 breast cancerxenografts was not significantly inhibited by treatment with NanoPac™ onthe day of evaluation but there was a significant difference on Days 19and 21. Additionally, there were two tumor-free animals through day 49(data not shown). Growth of the human MDA-MB-231 breast cancerxenografts was significantly inhibited by treatment with paclitaxelwhich was more effective than the NanoPac™ treatment on Days 15 and22-36. On Day 49 there were three tumor-free animals in the IVpaclitaxel treated group and two tumor-free animals in the IT NanoPac™treated group. Growth of the human MDA-MB-231 breast cancer xenograftswas significantly inhibited by treatment with docetaxel and NanoDoce™ OnDay 49 there were ten tumor-free animals in the IV docetaxel treatedgroup and eight tumor-free animals in the IT NanoDoce™ treated group.

Example 6. MX-1 Human Breast Cancer Xenograft Studies

The objective of this study was to determine the response of thesubcutaneously (SC)-implanted MX-1 human breast cancer xenografts totreatment with paclitaxel particles (referred to as NanoPac™) anddocetaxel particles (referred to as NanoDoce™). Test articles were asshown in Table 10:

TABLE 10 Dose Dose Dose Conc. Volume Group Test Article Vehicle (mg/kg)(mg/mL) (mL/kg) 1 Vehicle 0.1% T80/99.9% Sodium Chloride for 0 0 0.063*mL Injection USP (0.9%) 2 NanoPac ™ 0.1% T80/99.9% Sodium Chloride for100 40 0.063* mL Injection USP (0.9%) 3 NanoDoce ™ 0.2% T80/2%ethanol/18% PBS/79.8.% 100 40 0.063* mL Sodium Chloride for InjectionUSP (0.9%)** 4 Docetaxel 7.5% ethanol/7.5% T80/85% Sodium 30 3    10mL/kg Chloride for Injection USP (0.9%) 5 Paclitaxel 12.5% ethanol/12.5%Cremophor EL/ 30 3    10 mL/kg 75% Sodium Chloride for Injection USP(0.9%) *Based on a 25 g mouse **Due to a calculation error, the Vehicleformulation for NanoDoce was incorrectly described in the Protocol(Appendix A) as 0.267% T80/2.67% ethanol/23.71% PBS/73.33% saline(0.9%).

Dose Formulations:

NanoPac™:

1) Add 0.75 mL of the 1% T80 reconstitution solution into the vial ofNanoPac (306 mg/vial). Vigorously hand shake with inversion for 1minute. 3) Immediately after shaking, add 6.9 mL of 0.9% Sodium Chloridefor Injection USP to the vial to make a 40 mg/mL suspension (7.65 mL)and hand shake the vial for another 1 minute. After mixing, allow thesuspension to sit undisturbed for 5 minutes to reduce entrapped air andfoam.

Handling: Room temperature.

Stability: Dose formulations will be maintained at room temperature andconsidered stable for 8 hours.

NanoDoce™:

1) Add 1.0 ml of the 1% T80/10% ethanol/89% PBS reconstitution solutioninto the vial of NanoDoce particles (200 mg/vial). 2) Vigorously handshake the vial with inversions for 1 minute. 3) Immediately aftershaking, add 2 ml of 0.9% Sodium Chloride for Injection USP (0.9%) tothe vial and hand shake the vial for another 1 minute. 4) Add anadditional 2 ml of Sodium Chloride for Injection USP (0.9%) to the vialand continue hand mixing with inversion for 1 minute. Final volume 5 mLof 40 mg/mL NanoDoce suspension. After mixing, allow the suspension tosit undisturbed for at least 5 minutes to reduce entrapped air and foam.

Handling: Room temperature.

Stability: Dose formulations will be maintained at room temperature andconsidered stable for 8 hours.

Paclitaxel: 1) Add bulk paclitaxel to the clinical formulation ofpaclitaxel (6 mg/mL in 50% ethanol:50% Cremophor EL) to make a 12 mg/mLsolution. Vortex as needed. 2) Add Sodium Chloride for Injection USP(0.9%) to make a 3 mg/mL solution of paclitaxel. Vortex to mix.

Handling: Warm water.

Stability: Inject within 20 minutes of formulation.

Docetaxel:

1) Add Sodium Chloride for Injection USP (0.9%) to make a 3 mg/mLsolution of docetaxel (20 mg/mL in 50% ethanol:50% T80). Vortex to mix.

Test System:

Species & Strain: Mouse; NCr-nu/nu

Supplier: Charles River Laboratories

Number on Study: Females—50

Tumor Model:

A total of 88 female NCr-nu/nu mice were implanted with MX-1 breasttumor fragments from an in vivo passage subcutaneously (SC) on the rightflank. The day of tumor cell implantation was designated as Day 0.Individual tumors of 50 animals grew to 100-198 mg in weight (100-198mm³ in size) on Day 10 after tumor cell implantation, the day oftreatment initiation/Stage Day (SD). Fifty animals were assigned toGroups 1-5 such that the mean tumor weights in all five groups on Day 10were 139.9 to 141.9 mg (median tumor weights were 135 or 144 mg).

TABLE 11 No. Dose of Dose Route/ Dose Conc. Dose Total Dose Group miceTest Article Schedule (mg/kg) (mg/mL) Volume Delivered (mg) 1 10 VehicleIT*/qwk × 3 — — 0.063 mL 0 2 10 NanoPac ™ IT/qwk × 3 100 40 0.063 mL7.56 3 10 NanoDoce ™ IT/qwk × 3 100 40 0.063 mL 7.56 4 10 DocetaxelIV/qwk × 3 30 3 10 mL/kg 2.25 (0.10/10) 5 10 Paclitaxel IV/qwk × 3 30 310 mL/kg 2.25 (0.10/10) *IT = Intratumoral: use six needle tracks pertumor (27G ½ inch needle), inject one sixth of the volume as a slowbolus in each track, wait ~15 seconds then remove the needle slowly,space tracks over the surface of the tumor.

The NanoPac™ and NanoDoce™ are suspensions. Prior to filling eachsyringe and between the injections of each animal, the test article wasgently swirled/inverted. Animals were observed once daily for mortalityand moribundity. Adverse signs were documented by exception only. Bodyweights were collected twice weekly beginning on SD. Tumor measurementswere taken twice weekly beginning on SD, using a digital caliper. Lengthand width were measured for each tumor. Tumor volume was determinedusing the formula for an ellipsoid sphere: Length× Width 2/2=Volume(mm³). This formula was also used to calculate tumor weight, assumingunity density (1 mm³=1 mg). Limit of tumor detection is 32 mg (4×4 mm).The experiment lasted for 63 days from the day of tumor implant. Animalswhose weight decreased more than 30% from the weight on the first day oftreatment or whose tumor reached 4,000 mg in weight, ulcerated, orsloughed off was euthanized prior to study termination. Mean tumorvolumes for each treatment are shown in FIG. 4. More than 50% of animalsin groups 1-2 were lost due to tumor ulceration, death, or large tumorsize by day 26, and thus the mean data for these groups is notpresented.

Results

The human MX-1 breast cancer xenografts in the vehicle-treated controlgroup (Group 1) grew in all 10 mice to a mean tumor weight of 1,153 mgon Day 24 (n=9; the last day of treatment). There was one death (Day 21)and one moribund animal euthanized (Day 25) with a mean body weight lossof 1.06% (0.26 g, Day 14). Tumor inhibition was only compared until Day38 when there were 6 animals in the control group.

There were two animal deaths (Day 17 and 28) with three IT treatments(weekly) of NanoPac™ at a dose of 100 mg/kg (i.e., Group 2) and <1% meanbody weight loss. The animal death on Day 17 occurred after the ITinjection. The repeated NanoPac™ treatment did not significantly inhibitthe growth of the MX-1 breast cancer xenografts [mean tumor weight of1,096 mg on Day 24 (n=9) corresponding to a % difference of −4.9%relative to the vehicle-treated control (p>0.05)] The nadir of responseoccurred on Day 38 with a % difference of −14.1% which was notsignificantly different than the vehicle-treated control group (p>0.05).

There were two animal deaths (Day 17 and 24) during the three ITtreatments (weekly) of NanoDoce™ at a dose of 100 mg/kg (i.e., Group 3)and no mean body weight loss. The animal deaths on Days 17 and 24occurred after the IT injection. The repeated NanoDoc™ treatmentsignificantly inhibited the growth of the MX-1 breast cancer xenografts[mean tumor weight of 449 mg on Day 24 (n=9) corresponding to a %difference of −61.0% relative to the vehicle-treated control (p<0.01)].The tumor growth inhibition progressed to the nadir on Day 38 with a %difference of −90.1% (p<0.001); the mean tumor volume continued todecrease until Day 56. The % difference was significant starting on Day17 until no comparison was possible. There were four complete tumorregressions with the animals remaining tumor-free at the end of thestudy.

There were two animals euthanized for body weight loss >30% (Day 24 and31) following the three IV treatments (weekly) of docetaxel at a dose of30 mg/kg (i.e., Group 4) with a mean body weight loss of 24.9% (5.90 g,Day 31). The repeated docetaxel treatment significantly inhibited thegrowth of the MX-1 breast cancer xenografts [mean tumor weight of 0.9 mgon Day 28 (n=9) corresponding to a % difference of −99.9% relative tothe vehicle-treated control (p<0.001)]. The % difference was significantstarting on Day 14 until no comparison was possible. Complete tumorinhibition was observed on Day 31 when all nine animals had completelyregressed tumors. The eight surviving animals remained tumor-free at theend of the study on Day 63. The docetaxel treated animals' tumor growthinhibition was significantly different than the NanoDoce™ treatedanimals' from Days 14-52 but were not different from Day 56-63.

Three IV treatments (weekly) with paclitaxel at a dose of 30 mg/kg(i.e., Group 5) were tolerated without animal deaths and a mean bodyweight loss of 2.44% (0.59 g, Day 14). The paclitaxel treatmentsignificantly inhibited the growth of the MX-1 breast cancer xenografts[mean tumor weight of 309 mg on Day 24 (n=10) corresponding to a %difference of −73.2% relative to the vehicle-treated control (p<0.001)].The nadir of response occurred on Day 31 with a % difference of −86.0%.The % difference was significant starting on Day 14 until no comparisonwas possible. The paclitaxel treated animals' tumor growth inhibitionwas significantly different than the NanoPac™ treated animals' from Days14-38 (the last day any mice remained in the NanoPac™ treated group.

Discussion:

There were two animals dead, moribund, or euthanized for weight loss ineach group except for Paclitaxel treatment. Treatment with paclitaxelwas tolerated without animal deaths. Growth of the human MX-1 breastcancer xenografts was significantly inhibited by IT treatment withNanoDoce™ which had four tumor-free surviving animals. Growth of thehuman MX-1 breast cancer xenografts was inhibited by IV treatment withdocetaxel which had eight tumor-free surviving animals and wassignificantly different than NanoDoce™ treatment except at the end ofthe study (Days 56-63). Growth of the human MX-1 breast cancerxenografts was not significantly inhibited by treatment with NanoPac™.Growth of the human MX-1 breast cancer xenografts was significantlyinhibited by treatment with paclitaxel which was more effective than theNanoPac™ treatment.

Example 7. JIMT-1 Human Breast Cancer Xenograft Studies

The objective of this study was to determine the response of thesubcutaneously (SC)-implanted JIMT-1 human breast cancer xenografts totreatment with paclitaxel particles (referred to as Nanotax™). Testarticles were as follows:

Procedures:

-   -   100 CR female CB.17 SCID mice were injected with 1×10⁷ JIMT-1        tumor cells in 50% Matrigel sc in flank.    -   Cell Injection Volume was 0.1 mL/mouse.    -   Age at Start Date: 8 to 12 weeks.    -   Performed a pair match when tumors reached an average size of        100-150 mm³, and began treatment. Test articles were as shown in        Table 12:

TABLE 12 Regimen 1 Gr. N Agent mg/kg Route Schedule 1^(#) 10 vehicle —itu qwk × 5 2 10 Nanotax 37.5 itu qwk × 1 3 10 Nanotax 12.5 itu qwk × 54 10 Nanotax 37.5 itu qwk × 5 5 10 paclitaxel 30 iv qwk × 3 ^(#)ControlGroup paclitaxel = paclitaxel 5% Ethanol: 5% Cremophor EL in D5W Nanotax= Nanotax in 0.1% w/v Polysorbate 80 in Saline vehicle = 0.1% w/vPolysorbate 80 in Saline Dosing volume = 2.5 mL/kg (0.050 mL/20 gmouse). Adjust volume accordingly for body weight. For Group 5, IVpaclitaxel, dosing volume was 10 mL/kg (0.20 mL/20 g mouse).

-   -   Body Weight: 5/2 (measure 5 days, off for 2 day) then biwk to        end    -   Any individual animal with a single observation of >than 30%        body weight loss or three consecutive measurements of >25% body        weight loss was euthanized.    -   Endpoint TGD. Animals were monitored individually. The endpoint        of the experiment was a tumor volume of 1000 mm³ or 60 days,        whichever came first. When the endpoint is reached, the animals        were euthanized.

Tumor growth inhibition was evaluated on D29 (the last day of dosing foranimals receiving 5 admins, G1, 3, 4) at 1000 mm³ and the study ended onD50 with a revised tumor volume endpoint of 600 mm³ for overall survivalanalysis.

The tumors were then collected from the 3 best responders in each groupon day 50 for analysis to determine paclitaxel concentration in thetumor tissue. The timing of the analysis for each group relative to thelast day of drug administration is as follows:

-   -   21 days after last dose for groups 1, 3 and 4;    -   49 days for group 2; and    -   35 days for group 5.

These studies showed that Nanotax (NanoPac) was not significantly activein the JIMT-1 breast carcinoma model. Treatment of animals with fivedoses of Nanotax (NanoPac) resulted in small survival extensions and anincreased number of study survivors when compared to vehicle-treatedcontrol animals. Results for all Nanotax (NanoPac)-treated animals werewell below the 60% TGI threshold indicative of potential therapeuticactivity. Paclitaxel produced significant TGI and significantly improvedsurvival extension in this study. All treatments were well-tolerated.

Tissue processing was carried out as follows:

-   -   1. Control tumor tissue and tumor tissue samples were weighed        and three volumes of water were added to each to yield samples        with a dilution factor of 4.    -   2. Tumor tissues were homogenized with a homogenizer        FastPrep®-24 using a speed of 4.0 msec for 100 sec.    -   3. 50 μL of control tumor tissue homogenate was added to        separate wells of a 96 well plate for STDs, QC samples and        blanks.    -   4. To the control tumor tissue homogenates, 50 μL of the        appropriate solution was added for STDs and QC samples (50 μL of        diluent was added for blanks)    -   5. For tumor tissue homogenate study samples, 50 μL of each        sample was added to separate well of the 96-well plate and 50 μL        aliquot of diluent was added to each tissue sample.    -   6. To all samples, 200 μL of internal standard (200 ng/mL        Warfarin) solution in ACN was added.    -   7. The plate was vortexed vigorously for 10 minutes and then        centrifuged for 10 minutes at 4000 rpm (Sorvall Legend X1R        centrifuge, Thermo Scientific) at 15° C.    -   8. After centrifugation, 100 μL aliquots of supernatant were        transferred to a new 96-well plate containing 100 μL of water in        each well.    -   9. The plate was vortexed for approximately 1 minute and then        aliquots were injected for LC/MS/MS analysis.

The results are shown in the Table 13:

Tumor Tissue Sample Concentrations Paclitaxel Concentration Group AnimalPart (ng/g) 1 1 2 BQL < 1 ng/g 1 5 2 BQL < 1 ng/g 1 8 2 BQL < 1 ng/g 2 22 15300 2 5 2 3800 2 10 2 655000 3 1 2 1290000 3 6 2 1800 3 7 2 680 4 22 13400 4 6 2 1210 4 9 2 263 5 1 2 BQL < 1 ng/g 5 4 2 BQL < 1 ng/g 5 7 2BQL < 1 ng/g BQL: Below the quantitation limit of 1 ng/g before thedilution factor was applied. Group 1: Vehicle (IT, qwk × 3) Group 2:Nanotax (37.5 mg/kg, IT, qwk × 1) Group 3: Nanotax (12.5 mg/kg, IT, qwk× 3) Group 4: Nanotax (37.5 mg/kg, IT, qwk × 3) Group 5: Paclitaxel (30mg/kg, IV, qwk × 3)

This data surprisingly demonstrates that paclitaxel particles (groups 2,3, and 4) persisted for very long time periods in the tumor.

Example 8

Female nude mice (NCr-nu/nu) with MDA-MB-231 breast tumor xenograftsimplanted on the right flank (PD-PB-04-2016; see above) wereadministered 100 mg/kg doses of NanoDoce® as intratumor injections onDays 13, 20 and 27 following tumor implant. On Day 61 following tumorimplant, four Group 3 animals (100 mg/kg NanoDoce IT qwk×3) animals werenecropsied and skin and underlying tissue at the tumor implant site wereobtained. Tissues were homogenized in water. Aliquots of the homogenatewere extracted with acetonitrile and extracts were analyzed by LC/UV/MS.A synthetic standard of docetaxel was used to compare the retention time(T_(R)), UV and fragmentation pattern to the unknown compound present inthe tissue samples. The analyses of tumor site tissue extracts indicatedthat docetaxel was present in two samples (Animals 3-7 and 3-8) of thefour tissues examined. The LC/UV/MS analyses showed that the retentiontime (T_(R)=16.6 min) and MS² spectra of the chromatographic peak withinthe extracted ion chromatogram (XIC) of m/z 807.7-808.7 obtained fromthe tissue extracts was similar to the authentic standard sample ofdocetaxel (T_(R)=16.16 min).

In summary, based on similarity of retention time and mass spectral datawith the authentic reference standard, it was confirmed that in two offour mouse breast tumor (MDA-MB-231) xenografts models administeredintratumoral NanoDoce®, the compound isolated from tumor implant sitetissue samples was docetaxel.

Example 9. Phase IIa Dose Escalation Trial of Paclitaxel Particles(NanoPac™) Focal Therapy for Prostate Cancer in Subjects UndergoingRadical Prostatectomy

Protocol Summary

In this open-label, dose rising, Phase IIa trial with an expanded cohortat the dose of paclitaxel particles as described herein determined tohave the best tolerability and safety profile, subjects with prostatecancer scheduled for prostatectomy will have paclitaxel particlesinjected under image guidance directly into the lobe of the prostatewith the dominant lesion four weeks prior to prostatectomy. The studywill include a dose escalation phase and a dose confirmation phase.

In the dose escalation phase, paclitaxel particle concentrations of 6,10, and 15 mg/mL in an injection volume of up to 20% of the lobe of theprostate containing the dominant lesion will be studied in cohorts ofthree, with cohorts enrolled sequentially starting at the lowestconcentration. Following Data Safety Monitoring Board (DSMB) review ofthe cohort data the next cohort may begin enrolling, an additional threeat the current dose may be enrolled, or if the first dose does notprovide adequate safety and tolerability the study may be halted. Thedose determined to be the most suitable for further evaluation, definedas the highest dose with an acceptable safety and tolerability profileas determined by the DSMB, will enroll additional subjects to provide acohort of 12 subjects at that dose level.

Tumor volume and serum prostate-specific antigen (PSA) will bedetermined prior to paclitaxel particle injection. Pharmacokineticsamples, PSA, and ejaculate will be collected in the interval betweeninjection and prostatectomy. Imaging with multiparametric MRI (mpMRI)will be performed two to three weeks prior to paclitaxel particle (alsoreferred to as NanoPac™) injection and prior to prostatectomy. Prostateand pelvic lymph nodes excised at prostatectomy will be evaluated.

Endpoints:

Primary endpoint: Safety and tolerability, as demonstrated by adverseevents (AE), changes in laboratory assessments, physical examinationfindings, and vital signs.

Secondary endpoints:

-   -   Concentration of paclitaxel in the systemic circulation        post-injection and prior to prostatectomy;    -   Presence of paclitaxel in the tumor within the prostate, the        ipsilateral lobe of the prostate, the contralateral lobe of the        prostate, and pelvic lymph nodes excised during prostatectomy;    -   Tumor response (change in image volume on mpMRI; histologic        evaluation via biopsy);    -   Presence of tumor cells in the tumor within the prostate, the        ipsilateral lobe of the prostate, the contralateral lobe of the        prostate, and pelvic lymph nodes excised during prostatectomy.        Population: Up to a maximum of 30 men at a single site with        adenocarcinoma of the prostate scheduled for radical        prostatectomy.        Description of Study Agent:

NanoPac™ (sterile paclitaxel particles) Powder for Suspension(“NanoPac”) for direct injection into the lobe of the prostatecontaining the dominant lesion at concentrations of 6, 10, 15 mg/mL inan injection volume of up to 20% of the lobe of the prostate containingthe dominant lesion.

Name and Description of Study Agent:

A formulation of particulate paclitaxel, identified as NanoPac™ (sterileparticulate paclitaxel) Powder for Suspension, is manufactured using aPrecipitation with Compressed Antisolvent (PCA) technique that employssupercritical carbon dioxide and acetone to generate paclitaxelnanoparticles within a well-characterized particle-size distribution.Following PCA, paclitaxel particles are filled into 60 mL Type 1, USP,clear-glass vials (306 mg/vial), each of which is closed with abromobutyl rubber stopper and aluminum crimp seal, and sterilized bygamma irradiation. Prior to administration at the hospital/clinic,paclitaxel particles will be reconstituted with 1% Polysorbate 80, NF in0.9% sodium chloride (saline) for Injection, USP, to form a suspension.The suspension will be further diluted with 0.9% sodium chloride forInjection, USP to achieve the final clinical formulation. Thisreconstitution and dilution will occur at the clinical site's pharmacy.The NanoPac concentration in the final clinical formulation will beeither 6 mg/mL, 10 mg/mL, or 15 mg/mL. The final concentration ofPolysorbate 80 is 0.1% in the 6 mg/mL suspension, 0.16% in the 10 mg/mLsuspension, and 0.25% in the 15 mg/mL suspension.

Importance of the Study:

Prostate cancer is the second most common cancer in men, second only tonon-melanoma skin cancer. Despite the high prevalence of the disease, itis a constantly-evolving area of medicine, presenting difficultdecisions for patients and healthcare providers. At present, treatmentfor prostate cancer consists primarily of either of two options: activesurveillance or radical whole-gland therapy. However, this dichotomyfails to reflect the heterogeneity of prostate cancer and the nuancedpatient experience. Due, in part, to the widespread adoption of PSA as ascreening tool, more men are being diagnosed with lower-risk,lower-grade cancer. Active surveillance may be an appropriate choice forsome of these men, as radical whole-gland therapy risks life-alteringconsequences, such as impotence and incontinence. This is supported bythe statistic that 49% of men undergoing radical prostatectomy are foundto have only insignificant or indolent cancer. Nonetheless, prostatecancer has the fourth highest mortality rate of any cancer, and 73% ofpatients initially enrolled on active surveillance who ultimatelyundergo prostatectomy are found to have a significant cancer. As such,active surveillance not only risks disease progression, but can bepsychologically distressing to patients.

Rationale:

This Phase IIa study will include patients with adenocarcinoma of theprostate scheduled to undergo a prostatectomy. The study design allowsfor a safety evaluation of direct injection of paclitaxel particles intothe lobe of the prostate containing the dominant lesion as focal therapyprior to prostatectomy. We hypothesize that direct injection ofpaclitaxel particles into the prostate will result in limited, if any,systemic exposure to paclitaxel and should therefore result in onlylow-grade and transitory AE.

Direct injection, as opposed to intravenous (IV) administration, ofpaclitaxel particles would allow for higher concentrations of drug totarget local disease with reduced systemic toxicity. Intraprostaticpaclitaxel particles are expected to be more effective than IVpaclitaxel due to prolonged intraprostatic residence and dissolution,resulting in continuous and greater paclitaxel concentrations in thetumor site. Subjects will have NanoPac™ injected under magneticresonance imaging-transrectal ultrasound fusion (MR-TRUS) guidancedirectly into the lobe of the prostate containing the dominant lesion.Four weeks after injection of NanoPac™, the patient will undergo radicalprostatectomy.

Prior to study entry, subjects will undergo ultrasound-guided prostatebiopsy to diagnose and stage prostate cancer. This biopsy will be usedto identify the dominant lesion, which is defined as the lesion with thehighest Gleason score. The ultrasound performed during this biopsy willalso be used to calculate the volume of the entire prostate. The volumeof the lobe of the prostate containing the dominant lesion will bedetermined by calculating 50% of the total prostate volume as determinedby ultrasound at the time of pre-study biopsy. The study will include adose escalation phase and a dose confirmation phase.

-   -   Dose escalation: NanoPac™ concentrations of 6, 10, and 15 mg/mL        in an injection volume of up to 20% of the lobe of the prostate        containing the dominant lesion will be studied. Cohorts will be        enrolled sequentially starting at the lowest concentration. Each        cohort will have a planned minimum of three subjects. Data from        the first three subjects in a cohort will be reviewed and        evaluated by the DSMB. The outcome will be to determine whether        to a) escalate to the next dose; b) add three additional        subjects to the current dose; or c) expand the previous dose by        three subjects.    -   Dose confirmation phase: Once the dose deemed appropriate for        expansion and further evaluation has been determined, subjects        will be enrolled to that dose to provide a total of 12 subjects        dosed at that level. If two doses are similar, subjects will be        enrolled to each of the doses; however, the total number of        subjects treated will remain 12.        Standard of Care Study Procedures

The subjects being enrolled to this study will be undergoing a scheduledprostatectomy. Routine work-up prior to surgery, and the surgery itself,are considered standard of care. Follow-up to surgery will be perstandard of care. Serum PSA will be determined prior to paclitaxelparticle injection and weekly during the interval between paclitaxelparticle injection and prostatectomy.

Imaging with ultrasound and mpMRI will have been conducted prior tostudy participation as part of the routine evaluation and confirmationof adenocarcinoma. These results will serve as the pre-treatment (orBaseline) data, prior to paclitaxel particle injection; anotherstudy-specific mpMRI will then be conducted within three days prior toprostatectomy, and ultrasound will be performed as part of theprostatectomy procedure.

In the interval between paclitaxel particle injection and prostatectomy,starting one week after injection, ejaculate will be collected on aweekly basis and evaluated for determination of paclitaxelconcentration.

On the day of prostatectomy, after anesthesia has been administered, abiopsy will be performed. Immediately following biopsy, under the sameanesthesia, subjects will have the scheduled prostatectomy and,following surgery, samples will be taken from the excised tissue(prostate and lymph nodes) for evaluation of paclitaxel concentrationand for assessment of tumor cells, as described previously.

Example 10. Phase IIa Trial Evaluating the Safety of IntratumoralInjection of Paclitaxel Particles in Subjects with Locally AdvancedPancreatic Adenocarcinoma

In this open-label Phase IIa trial, subjects with locally advancedpancreatic adenocarcinoma located in the tail or body of the pancreaswill have completed at least one course of chemotherapy as part ofStandard of Care (SOC). Once there is sufficient hematologic recovery,subjects will receive ITU paclitaxel particles (i.e., NanoPac™) viaendoscopic ultrasound-guided direct injection. Subjects will be followedfor overall survival (OS), progression-free survival (PFS), CA-19-9levels, carcinoembryonic antigen (CEA) levels, reduction in pain, andtumor response to therapy (as shown by imaging).

Subjects will be enrolled in sequential, escalating cohorts ofpaclitaxel particles at concentrations of 6, 10, or 15 mg/mL injecteddirectly into the tumor within the pancreas at up to 20% tumor volume(with a maximum of 5 mL volume being administered to any subject). Thestudy will include a dose escalation phase and a dose confirmationphase.

Based on these tumor volume calculations, an amount of paclitaxelparticles equal to 20% of tumor volume, not to exceed 5 mL in anysubject, may be injected into the tumor.

The paclitaxel particles for use in the study (referred to as NanoPac™)are manufactured by CritiTech, Inc. (Lawrence, Kans.) using aPrecipitation with Compressed Antisolvent (PCA) technique that employssupercritical carbon dioxide and acetone to generate paclitaxelnanoparticles within a well-characterized particle-size distribution.Prior to administration at the hospital/clinic, the paclitaxel particlepowder in vial is suspended with Sterile Reconstitution Solution (1%Polysorbate 80, NF in 0.9% Sodium Chloride for Injection, USP) and thenfurther diluted with 0.9% Sodium Chloride for Injection, USP, resultingin a 6, 10, or 15 mg/mL NanoPac™ suspension.

Introduction of the particulate paclitaxel suspension into the site ofthe malignancy is hypothesized to create a depot of paclitaxel withinthe affected area (in this case, the pancreatic tumor), slowly releasingthe paclitaxel from the particles into the tumor space and thereforeresulting in prolonged paclitaxel exposure at the site of malignancy.Reduced clearance from the pancreas should result in lower systemiclevels of paclitaxel, further limiting systemic toxicity.

The Phase IIa study will be carried out to evaluate the safety andtolerability of paclitaxel particles in up to 30 patients who will beundergoing chemotherapeutic treatment for locally advanced pancreaticadenocarcinoma located in the tail or body of the pancreas. In thisclinical trial, paclitaxel particles will be administered directly intothe pancreas via endoscopic ultrasound-guided fine needle injection(EUS-FNI).

The drug product is contained in a clear 60 mL Type 1, USP, clear-glassvial (306 mg/vial) as a powder fill of particulate paclitaxel, closedwith a butyl rubber stopper and aluminum crimp seal, and sterilized bygamma irradiation. For clinical administration, the paclitaxel particlepowder in vial is suspended with Sterile Reconstitution Solution (1%Polysorbate 80, NF in 0.9% Sodium Chloride for Injection, USP) and thenfurther diluted with varying volumes of 0.9% Sodium Chloride forInjection, USP. The result is a 6, 10, or 15 mg/mL NanoPac suspensioncontaining 0.1, 0.16, or 0.25% Polysorbate 80, NF, respectively, in 0.9%Sodium Chloride for Injection, USP.

TABLE 14 Components of NanoPac ® (sterile nanoparticulate paclitaxel)Powder for Suspension and final formulation Reference to QualityComponent Function Standards Amount Components of NanoPac, SterilePowder for Suspension Paclitaxel^(a) Active USP 306.0 mg/vialAcetone^(b) Processing HPLC Grade ^(b) fluid Carbon Processing ISBTBeverage ^(b) Dioxide^(b) Grade 99.9% fluid pure Components of NanoPac,Final Formulation Paclitaxel^(a) Active USP 306.0 mg/vial PolysorbateSurfactant NF 0.1% (6 mg/mL NanoPac) 80 0.16% (10 mg/mL NanoPac) 0.25%(15 mg/mL NanoPac) 9% Sodium Suspension USP 51 mL (6 mg/mL NanoPac)Chloride for solution 30.6 mL (10 mg/mL Injection NanoPac) 20.4 mL (15mg/mL NanoPac) ^(a)= In nanoparticulate form. ^(b)= Removed duringprocessing.

Endpoints:

Primary endpoint: Safety and tolerability as demonstrated by adverseevents (AE), changes in laboratory assessments, physical examinationfindings and vital signs.

Secondary endpoints:

-   -   Concentration of paclitaxel in the systemic circulation        post-injection (as determined by PK analysis);    -   Tumor response (RECIST as per Eisenhauer et al. 2009);    -   Reduction in pain (as measured by the visual analog scale        [VAS]);    -   Change in tumor marker CA19-9;    -   Change in tumor marker CEA.

Population:

Up to 30 subjects with locally advanced pancreatic adenocarcinomalocated in the tail or body of the pancreas.

We claim:
 1. A method for treating a solid tumor, comprising administering to a subject with a solid prostate tumor an amount effective of a composition comprising chemotherapeutic particles to treat the prostate tumor, wherein the composition is directly injected into the prostate tumor, wherein the chemotherapeutic particles comprise at least 95% paclitaxel or a pharmaceutically acceptable salt thereof, and wherein the chemotherapeutic particles have a specific surface area (SSA) of at least 10 m²/g, wherein the chemotherapeutic particles include both agglomerated chemotherapeutic particles and non-agglomerated chemotherapeutic particles.
 2. The method of claim 1 wherein the composition consists of the chemotherapeutic particles and a pharmaceutically acceptable carrier.
 3. The method of claim 2, wherein the carrier is an aqueous liquid carrier.
 4. The method of claim 3 wherein the aqueous liquid carrier is saline.
 5. The method of claim 1, wherein the composition is a suspension.
 6. The method of claim 1, wherein the chemotherapeutic particles have an SSA of between about 10 m²/g and about 50 m²/g.
 7. The method of claim 1, wherein the chemotherapeutic particles have a SSA of at least 18 m²/g.
 8. The method of claim 7, wherein the chemotherapeutic particles have a mean bulk density between about 0.050 g/cm³ and about 0.12 g/cm³.
 9. The method of claim 7, wherein the chemotherapeutic particles have a SSA of between about 22 m²/g and about 40 m²/g.
 10. The method of claim 1, wherein the chemotherapeutic particles have a mean particle size number of between about 0.4 μm and about 1.2 μm.
 11. The method of claim 1, wherein the chemotherapeutic particles are present in a suspension further comprising a pharmaceutically acceptable aqueous carrier.
 12. The method of claim 11, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of between about 0.01% v/v and about 1.5% v/v.
 13. The method of claim 11, wherein the paclitaxel, or pharmaceutically acceptable salt thereof, is present in the suspension at a concentration between about 1 mg/ml and about 40 mg/ml.
 14. The method of claim 2, wherein the chemotherapeutic particles are present in a suspension further comprising a pharmaceutically acceptable aqueous carrier.
 15. The method of claim 6, wherein the chemotherapeutic particles are present in a suspension further comprising a pharmaceutically acceptable aqueous carrier.
 16. The method of claim 7, wherein the chemotherapeutic particles are present in a suspension further comprising a pharmaceutically acceptable aqueous carrier.
 17. The method of claim 9, wherein the chemotherapeutic particles are present in a suspension further comprising a pharmaceutically acceptable aqueous carrier.
 18. The method of claim 10, wherein the chemotherapeutic particles are present in a suspension further comprising a pharmaceutically acceptable aqueous carrier.
 19. The method of claim 14, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of between about 0.01% v/v and about 1.5% v/v.
 20. The method of claim 15, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of between about 0.01% v/v and about 1.5% v/v.
 21. The method of claim 16, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of between about 0.01% v/v and about 1.5% v/v.
 22. The method of claim 17, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of between about 0.01% v/v and about 1.5% v/v.
 23. The method of claim 18, wherein the suspension further comprises a polysorbate, wherein the polysorbate is present in the suspension at a concentration of between about 0.01% v/v and about 1.5% v/v.
 24. The method of claim 2, wherein the chemotherapeutic particles have a mean particle size number of between about 0.4 μm and about 1.2 μm.
 25. The method of claim 6, wherein the chemotherapeutic particles have a mean particle size number of between about 0.4 μm and about 1.2 μm.
 26. The method of claim 9, wherein the chemotherapeutic particles have a mean particle size number of between about 0.4 μm and about 1.2 μm. 